Recombinant poliovirus for the treatment of cancer

ABSTRACT

The present invention is directed to non-pathogenic, oncolytic, recombinant polioviruses for the treatment of various forms of malignant tumors. The recombinant polioviruses of the invention are those in which the internal ribosomal entry site (IRES) of the wild type poliovirus was exchanged with the IRES of other picornaviruses, and optionally P1, P3 or the 3′NTR thereof was exchanged with that of poliovirus Sabin type. More particularly, the present invention is directed to the administration of the non-pathogenic, oncolytic, recombinant poliovirus to the tumor directly, intrathecally or intravenously to cause tumor necrosis. The method of the present invention is particularly useful for the treatment of malignant tumors in various organs, such as: breast, colon, bronchial passage, epithelial lining of the gastrointestinal, upper respiratory and genito-urinary tracts, liver, prostate and the brain. Astounding remissions in experimental animals have been demonstrated for the treatment of malignant glioblastoma multiforme, an almost universally fatal neoplasm of the central nervous system.

[0001] The invention was made with Government support under No.AI32100-07 and AI39485 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

[0002] The present invention is directed to non-pathogenic, oncolytic,recombinant polioviruses for the treatment of various forms of malignanttumors. More particularly, the present invention is directed to theadministration of the non-pathogenic, oncolytic, recombinant poliovirusto the tumor directly, intrathecally or intravenously to cause tumornecrosis. The method of the present invention is particularly useful forthe treatment of malignant tumors in various organs, such as: breast,colon, bronchial passage, epithelial lining of the gastrointestinal,upper respiratory and genito-urinary tracts, liver, prostate and thebrain. Astounding remissions in experimental animals have beendemonstrated for the treatment of malignant glioblastoma multiforme, analmost universally fatal neoplasm of the central nervous system.

BACKGROUND OF THE INVENTION

[0003] Known Methods of Treatment

[0004] It has been known that malignant tumors result from theuncontrolled growth of cells in an organ. The tumors grow to an extentwhere normal organ function may be critically impaired by tumorinvasion, replacement of functioning tissue, competition for essentialresources and, frequently, metastatic spread to secondary sites.Malignant cancer is the second leading cause of mortality in the UnitedStates.

[0005] Up to the present, the methods for treating malignant tumorsinclude surgical resection, radiation and/or chemotherapy. However,numerous malignancies respond poorly to all traditionally availabletreatment options and there are serious adverse side effects to theknown and practiced methods. There has been much advancement to reducethe severity of the side effects while increasing the efficiency ofcommonly practiced treatment regimens. However, many problems remain,and there is a need to search for alternative modalities of treatment.The search is particularly urgent for primary malignant tumors of thecentral nervous system. Brain tumors, especially glioblastomas, remainone of the most difficult therapeutic challenges. Despite theapplication of surgery, radiotherapy and chemotherapy, alone and incombination, glioblastomas are almost always fatal, with a mediansurvival rate of less than a year and 5-year survival rates of 5.5% orless. None of the available therapeutic modes has substantially changedthe relentless progress of glioblastomas.

[0006] Systematic studies of patients who were diagnosed with malignantglioma and underwent surgery to wholly or partially remove the tumorwith subsequent chemotherapy and/or radiation showed that the survivalrate after 1 year remains very low, particularly for patients who areover 60 ears of age. Leibel, S. A., et al., Cancer, 35:1551-1557 (1975);Walker, M. D., et al., J. Neurosurg., 49:333-343 (1978); Chang, C. H.,et al., Cancer, 52:997-1007 (1983). Malignant gliomas have proven to berelatively resistant to radiation and chemotherapeutic regimens. Bloom,H. J. G., Int. J. Radiat. Oncol. Biol. Phys., 8:1083-1087 (1982). Addingto the poor prognosis for malignant gliomas is the frequent tendency forlocal recurrence after surgical ablation and adjunctradiation/chemotherapy. Choucair, A. K., et al., J. Neurosurg.,65:654-658 (1986).

[0007] Treatment of Cancer with Viruses

[0008] In recent years, there have been proposals to use viruses for thetreatment of cancer: (1) as gene delivery vehicles, Miller, A. D.,Nature, 357:455-460 (1992); (2) as direct oncolytic agents by usingviruses that have been genetically modified to lose their pathogenicfeatures, Martuza, R. L., et al., Science, 252:854-856 (1991); or (3) asagents to selectively damage malignant cells using viruses which havebeen genetic engineered for this purpose, Bischoff, J. R., et al.,Science, 274:373-376 (1996).

[0009] Examples for the use of viruses against malignant gliomas includethe following.

[0010] Herpes Simplex Virus dlsptk (HSVdlsptk), is a thymidine kinase(TK)-negative mutant of HSV. This virus is attenuated for neurovirulencebecause of a 360-base-pair deletion in the TK gene, the product of whichis necessary for normal viral replication. It has been found thatHSVdlsptk retains propagation potential in rapidly dividing malignantcells, causing cell lysis and death. Unfortunately, all defective herpesviruses with attenuated neuropathogenicity have been linked with serioussymptoms of encephalitis in experimental animals. Wood, M. J. A., etal., Gene Therapy, 1:283-291 (1994). For example, in mice infectedintracerebrally with HSVdlsptk, the LD₅₀ ^(Ic) (intracranialadministration) is 10⁶ pfu, a rather low dose. This limits the use ofthis mutant HSV. Markert, J. M., et al., Neurosurgery, 32:597-603(1993). Other mutants of HSV have been proposed and tested.Nevertheless, death from viral encephalitis remains a problem. MinetaT., et al., Nature Medicine, 1:938-943 (1995); Andreansky, S., et al.,Cancer Res., 57:1502-1509 (1997).

[0011] Another proposal is to use retroviruses engineered to contain theHSV tk gene to express thymidine kinase which causes in vivophosphorylation of nucleoside analogs, such as gancyclovir or acyclovir,blocking the replication of DNA and selectively killing the dividingcell. Izquierdo, M., et al., Gene Therapy, 2:66-69 (1995) reported theuse of Moloney Murine Leukemia Virus (MoMLV) engineered with aninsertion of the HSV tk gene with its own promoter. Follow-up ofpatients with glioblastomas that were treated with intraneoplasticinoculations of therapeutic retroviruses by MRI revealed shrinkage oftumors with no apparent short-term side effects. However, theexperimental therapy had no effect on short-term or long-term survivalof affected patients. Retroviral therapy is typically associated withthe danger of serious long-term side effects (e.g. insertionalmutagenesis).

[0012] Chen, S. H., et al., PNAS, USA, 91:3054-3057 (1994) reported thedirect injection of a recombinant into experimentally induced gliomas inathymic mice. ADV/RSV-TK is an adenovirus containing the HSV-tk geneunder transcriptional control of the rous sarcoma virus long terminalrepeat, followed by treatment with gancyclovir. The treatment causedtumor necrosis without apparent involvement of the cellular immuneresponse. The treated animals survived >50 days after tumor inoculationas contrasted with control tumor inoculated animals all of which diedafter 23 days. However, further long-term toxicity testing of neuronal,glial and endothelial cells is necessary to assess the potential ofgenetically engineered retroviruses for the treatment of cancers.

[0013] Recently, a novel strategy to use human pathogenic viruses forthe treatment of malignant disease was introduced. Adenovirus engineeredto selectively replicate within and destroy malignant cells expressing amodified p53 tumor suppressor offers an opportunity to target malignantcells without causing unwanted side effects due to virus propagation atextratumoral sites. Bischoff, J. R., et al., supra.

[0014] Similar systems have been developed to target malignancies of theupper airways, tumors that originate within the tissue naturallysusceptible to adenovirus infection and that are easy accessible.However, Glioblastoma multiforme, highly malignant tumors composed outof widely heterogeneous cell types (hence the denomination multIforme)are characterized by exceedingly variable genotypes and are unlikely torespond to oncolytic virus systems directed against homogeneous tumorswith uniform genetic abnormalities.

[0015] The Cells of the Central Nervous System

[0016] It is important to recognize that there are two classes of cellsin the brain, the neural cells (neurons) and the neuroglia cells (glia).Neurons process information received from the peripheral receptorsgiving rise to perception and memory. Motor commands are issued andtransmitted also by means of neurons to the various muscles of the body.There are nine times more glial cells than neurons. The glial cells havemultiple functions. They serve as the supporting elements; segregateneurons into disparate groups and produce myelin. Based on physiologicalcharacteristics, there are five major classes of glial cells:astrocytes, oligodendrocytes, microglia, ependymal cells, and Schwanncells. Kandel, E. R. and Schwartz, J. H., ed., Principles of NeuralScience, Chapter 2, pp. 14-23 Elsevier/North, Holland, 1981.

[0017] It is known that both the neurons and glial cells emerge from theneuroepithelium of the primitive neural tube. However, the timing andplace of the mechanisms that underlie the separation of neuronal andglial cell lines have been unsettled and controversial. In 1889, Hisproposed that the germinative epithelium consists of two classes ofprecursor cells: one that produces neurons and another that producesglial cells. Although disputed, this has proven to be correct. It isbelieved that glial cells are generated after all or a majority of theneurons destined for a given structure have been formed. Black, I., ed.Cellular and Molecular Biology of Neuronal Development, Chapter 2, pp.29-47, Plenum Press, New York, 1984.

[0018] The Poliovirus

[0019] Poliomyelitis is a disease of the central nervous system causedby infection with poliovirus. Poliovirus is a human enterovirus thatbelongs to the PIcornavIrIdae family and is classified into three stableserotypes. It is spherical, 20 nm in size, and contains a core of RNAcoated with a capsule consisting of proteins. It is transmitted throughthe mucosa of the mouth, throat or the alimentary canal. All threepoliovirus serotypes have been reported as causative agents of paralyticpoliomyelitis, albeit at different frequencies (type 1>type 2>type 3).

[0020] However, infection by poliovirus does not necessarily lead to thedevelopment of poliomyelitis. On the contrary, the majority ofinfections (98-99%) lead to local gastrointestinal replication of thevirus causing only mild symptoms, or no symptoms at all. Rarely doespoliovirus invade the CNS where it selectively targets spinal cordanterior horn and medullary motor neurons for destruction. Bodian, D.,in: Diseases of the Nervous System, Minckler, J. ed., McGraw-Hill, NewYork, pp.2323-2339 (1972).

[0021] The unusually restricted cell tropism of poliovirus leads tounique pathognomonic features. They are characterized by motor neuronloss in the spinal cord and the medulla, giving rise to the hallmarkclinical sign of poliomyelitis, flaccid paralysis. Other neuronalcomponents of the central nervous system as well as glial cellstypically escape infection. In infected brain tissue under theelectronmicroscope, severe changes are observed in motor neurons whereasno significant alterations are observed in the neuroglial components.Normal astrocyes and oligodendrocytes may be seen next to degenerateneurons or axons without evidence of infection or reaction. Bodian, D.,supra. The restricted tropism of poliovirus is not understood. Inaddition to the restricted cell and tissue tropism, poliovirus onlyinfects primates and primate cell cultures. Other mammalian speciesremain unaffected. Ren, R., et al., Cell, 63:353-362 (1990).

[0022] The isolation of poliovirus in 1908 led to intensive researchefforts to understand the mechanisms of infection. The earlier workrequired the use of monkeys and chimpanzees as animal models. Suchanimals with longer life cycles are very costly and difficult to use inresearch. The discovery of the human poliovirus receptor (PVR) alsoknown as CD155, the cellular docking molecule for poliovirus, led to thedevelopment of a transgenic mouse expressing the human poliovirusreceptor as a new animal model for poliomyelitis. The pathogenicity ofpoliovirus may be studied using the transgenic mice. Ren et al. (1990);Koike, S.,, et al., PNAS, USA, 88:951-955 (1991).

[0023] The early research efforts have also led to the development ofattenuated PV strains that lack neuropathogenic potential and soon weretested as potential vaccine candidates for the prevention ofpoliomyelitis. The most effective of these are the Sabin strains of type1, 2, and 3, of poliovirus developed by A. Sabin. Sabin & Boulger., Dev.Biol. Stand. 1:115-118 (1973). After oral administration of the liveattenuated strains of poliovirus (the Sabin strains) vaccine-associatedparalytic poliomyelitis has been observed in extremely rare cases. Theoccurrence of vaccine-associated paralytic polio has been correlatedwith the emergence of neurovirulent variants of the attenuated Sabinstrains after immunization. Minor, P. D., Dev. Biol. Stand., 78:17-26(1993).

[0024] In order to understand the invention, it is important also tohave an understanding of the structure of poliovirus.

[0025] All picornaviruses including enteroviruses, cardioviruses,rhinoviruses, aphthoviruses, hepatovirus and parechoviruses contain 60copies each of four polypeptide chains: VP1, VP2, VP3, and VP4. Thesechains are elements of protein subunits called mature “protomers”. Theprotomer is defined as the smallest identical subunit of the virus.Traces of a fifth protein, VP0, which is cleaved to VP2 and VP4 are alsoobserved. Together, these proteins form the shell or coat of poliovirus.

[0026] The picornaviral genome consists of a single strand ofmessenger-active RNA. The genomic messenger active RNA consists of a “+”strand which is polyadenylated at the 3′ terminus and carries a smallprotein, VPg, covalently attached to the 5′ end. The first picornaviralRNA to be completely sequenced and cloned into DNA was that of a type 1poliovirus. However, polioviruses lack a 5′m⁷GpppG cap structure, andthe efficient translation of RNA requires ribosomal binding that isaccomplished through an internal ribosomal entry site (IRES) within the5′ untranslated region (5′NTR).

[0027] The common organizational pattern of a poliovirus is representedschematically in FIG. 1, which comprises 5′NTR, P1, P2, P3 and 3′NTRwith a polyadenylated tail. The 5′NTR comprises 6 domains arbitrarilydesignated as I, II, III, IV, V, and VI. The IRES comprises domainsII-VI. P1 is the coding region for structural proteins also known as thecapsid proteins. P2 and P3 encode the non-structural proteins. Aschematic diagram of the six domains of the 5′NTR is represented in FIG.2.

[0028] In nature, three immunologically distinct poliovirus types occur:serotype 1, 2, and 3. These types are distinct by specific sequences intheir capsid proteins that interact with specific sets of neutralizingantibodies. All three types occur in different strains, and allnaturally occurring types and strains can cause poliomyelitis. They are,thus, neurovirulent. The genetic organization and the mechanism ofreplication of the serotypes are identical; the nucleotide sequences oftheir genomes are >90% identical. Moreover, all polioviruses, even theattenuated vaccine strains, use the same cellular receptor (CD155) toenter and infect the host cells; and they express the same tropism fortissues in human and susceptible transgenic animals.

[0029] The neuropathogenicity of poliovirus can be attenuated bymutations in the regions specifying the P1 and P3 proteins as well as inthe internal ribosomal entry site (IRES) within the 5′NTR. The Sabinvaccine strains of type 1, 2, and 3 carry a single mutation each indomain V of their IRES elements that has been implicated in theattenuation phenotype. Despite their effectiveness as vaccines, theSabin strains retain a neuropathogenic potential in animal models forpoliomyelitis. Albeit at a very low rate, they can cause the disease invaccinees.

[0030] Indeed, the single point mutations in the IRES element of eachSabin vaccine strain can revert in a vaccinee within a period of 36hours to several days. Overall, vaccine associated acute poliomyelitisoccurs in the United States at a rate of 1 in 530,000 vaccinees. Thepolioviruses isolated from vaccinated patients with poliomyelitis mayalso have mutations reverted in different positions of their genomes.Wimmer, E., et al., Ann.Rev.Gen., 27:353-436 (1993), Minor, P. D.,supra.

[0031] Recombinant Polioviruses

[0032] Chimeric polioviruses carrying heterologus IRES elements, whichhave lost their inherent neuropathogenic potential have been described.Gromeier, M., et al., Proc. Natl. Acad. Sci. USA, 93:2370-2375 (1996),incorporated herein by reference. It was found that the substitution ofthe cognate IRES of poliovirus with its counterpart from HumanRhinovirus type 2 (HRV2) eliminated the ability of the resultingchimera, PV1(RIPO) to grow within cells of neuronal derivation (FIGS. 3Aand B). The inventors also described the construction of andneurovirulence testing of a chimera carrying the P1 coding region forthe structural proteins derived from PV1(S), PV1(RIPOS) in addition tothe heterologous IRES originating from HRV2.

[0033] The non-pathogenic phenotype of PV1(RIPO) and PV1(RIPOS) wasdocumented in mice transgenic for the human poliovirus receptor, CD155tg mice. See Gromeier et al., supra. It was shown that non-pathogenicPV/HRV2 IRES chimeras are unable to cause the typical lesions of thespinal cord typical of poliomyelitis when injected intracerebrally intoCD155 tg mice. The non-pathogenic property of these constructs are nowshown in Cynomolgus monkeys (FIG. 3A). The non-human primates thatreceived intraspinal inoculations of PV1(RIPO) or PV1(RIPOS) remainedunaffected or developed transient, subtle pareses of one foot in anisolated case. Permanent neurological dysfunction or signs ofpoliomyelitic disease were not noticed in any of the treated monkeys.

[0034] Despite its inability to replicate efficiently within cells ofneuronal origin, it is now shown that PV1(RIPO) retained wild-typegrowth characteristics with an ability to lyse tumor cells in a panel ofrapidly dividing malignant cell types originating from humanmalignancies (FIGS. 10-17).

OBJECTIVES OF THE INVENTION

[0035] It is an objective of the present invention to developnon-neuropathogenic polioviruses for the treatment for various types ofcancer, in particular cancer of the central nervous system.

[0036] It is a further objective of the present invention to treatcancer cells by infecting them with a nonpathogenic poliovirus to causecancer cell lysis and death.

[0037] It is another objective of the present invention to developfurther novel poliovirus chimeras, which would be suitable for thetreatment of cancer.

[0038] It is a further objective of the present invention to developfurther novel poliovirus chimeras, which would be suitable for thetreatment and cure of gliomas, in particular glioblastomas.

LIST OF REFERENCES

[0039] 1. Leibel, S. A., et al., Cancer, 35:1551-1557 (1975).

[0040] 2. Walker, M. D., et al., J. Neurosurg., 49:333-343 (1978).

[0041] 3. Chang, C. H., et al., Cancer, 52:997-1007 (1983).

[0042] 4. Bloom, H. J. G., Int. J. Radiat. Oncol. Biol. Phys.,8:1083-1087 (1982).

[0043] 5. Choucair, A. K., et al., J. Neurosurg., 65:654-658 (1986).

[0044] 6. Miller, A. D., Nature, 357:455-460 (1992).

[0045] 7. Martuza, R. L., et al., Science, 252:854-856 (1991).

[0046] 8. Bischoff, J. R., Science, 274:373-376 (1996).

[0047] 9. Wood, M. J. A., et al., Gene Therapy, 1:283-29, (1994).

[0048] 10. Markert, J. M., et al., Neurosurgery, 32:597-603 (1993).

[0049] 11. Mineta T., et al., Nature Medicine, 1:938-943 (1995).

[0050] 12. Andreansky, S., et al., Cancer Res.,57:1502-1509 (1997).

[0051] 13. Izquierdo, M., et al., Gene Therapy, 2:66-69 (1995).

[0052] 14. Chen, S. H., et al., PNAS, USA, 91:3054-3057 (1994).

[0053] 15. Kandel, E. R. and Schwartz, J. H., ed. Principles of NeuralScience, Chapter 2, pp. 14-23 Elsevier/North, Holland, 1981.

[0054] 16. Black, I., ed. Cellular and Molecular Biology of NeuronalDevelopment, Chapter 2, pp. 29-47, Plenum Press, New York, 1984.

[0055] 17. Bodian, D., Diseases of the Nervous System, Chapter 170pp.2323-2339, McGraw Hill, New York.

[0056] 18. Ren, R.,, et al., Cell, 63:353-362 (1990).

[0057] 19. Koike, S., et al., PNAS, USA, 88:951-955 (1991).

[0058] 20. Sabin & Boulger, Dev. Biol. Stand., 1:115-118 (1973).

[0059] 21. Minor, P. D., Dev. Biol. Stand., 78:17-26 (1993).

[0060] 22. Wimmer, E., et al., Ann. Rev. Gen., 27:353-436 (1993).

[0061] 23. Gromeier, M., et al., Proc. Natl. Acad. Sci. USA,93:2370-2375 (1996).

[0062] 24. Sambrook, Fritsch and Maniatis, Molecular Cloning, ColdSpring Harbor Laboratory Press, N.Y. (1989).

[0063] 25. Wimmer, et al., U.S. Pat. No. 5,674,729.

[0064] 26. Omata, T.,et al., J. Virol., 58:348-358 (1986).

[0065] 27. WHO Technical Report Series No. 80 (1990).

[0066] 28. Kawamura, N., et al., J Virol., 63:1302-1309 (1989).

[0067] 29. Fogh, J., et al., J. Natl. Cancer. Inst., 59:221-226 (1997).

[0068] 30. Agol, et al., J. Virol., 63:4034-4038 (1989).

[0069] 31. LaMonica, N. and Rancaniello, V. R., J. Virol., 63:2357-2360(1989).

[0070] 32. Reed and Muench, Am. J. Hyg., 27:493-495 (1938).

SUMMARY OF THE INVENTION

[0071] According to the present invention, non-neuropathogenic,oncolytic, chimeric recombinant polioviruses have been engineered. Theoncolytic chimeric polioviruses comprise:

[0072] A recombinant poliovirus constructed from a poliovirus having a5′NTR region containing an internal ribosomal entry site (IRES), and thecoding sequences for structural proteins (P1), and for thenon-structural proteins (P2 and P3) and a 3′NTR selected from the groupconsisting of wild type serotype 1, serotype 2, and serotype 3, wherein

[0073] a. i. a part of the IRES of the poliovirus is substituted with apart of the IRES of Human Rhinovirus serotype 2 also having a 5′NTRregion containing an internal ribosomal entry site (IRES), the codingsequences of structural proteins (P1), and for the non-structuralproteins (P2 and P3) and a 3′NTR, or

[0074] ii. at least a part of the IRES of the poliovirus is substitutedwith at least a part of the IRES of a virus selected from the group ofpicornaviruses comprising Human Rhinovirus serotype 1, 3-100,coxsackievirus serotype B1-B6, human echovirus serotype 1-7, 9, 11-27,29-33, all of which also having a 5′NTR region containing an internalribosomal entry site (IRES), the coding sequences of structural proteins(P1), and for the non-structural proteins (P2 and P3) and a 3′NTR, andwherein

[0075] b. optionally, at least a part of the P1 of the poliovirus issubstituted respectively with at least a part of the P1 of a Poliovirus(Sabin), selected from the group consisting of PV1(S), PV2(S) andPV3(S);

[0076] c. optionally, at least a part of the P3 of the poliovirus issubstituted with at least a part of the P3 of Poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S) and PV3(S); and

[0077] d. optionally, at least a part of the 3′NTR of the wild typepoliovirus is substituted with at least a part of the entire 3′NTR ofpoliovirus (Sabin), selected from the group consisting of PV1(S),PV2(S), and PV3(S).

[0078] The invention is further directed to a therapeutic method oftreating malignant tumors comprising the steps:

[0079] A. Preparing a nonpathogenic recombinant poliovirus having a5′NTR region containing an internal ribosomal entry site (IRES), and thecoding sequences for structural proteins (P1), and for thenon-structural proteins (P2 and P3) and a 3′NTR selected from the groupconsisting of wild type serotype 1, serotype 2, and serotype 3, by

[0080] a. substituting at least a part of the IRES of the polioviruswith at least a part of the IRES of a virus selected from the group ofpicornaviruses comprising Human Rhinovirus serotype 1-100,coxsackievirus serotype B1-B6, human echovirus serotype 1-7, 9, 11-27,29-33, all of which also having a 5′NTR region containing an internalribosomal entry site (IRES), the coding sequences of structural proteins(P1), and for the non-structural proteins (P2 and P3) and a 3′NTR;

[0081] b. optionally substituting at least a part of the P1 of thepoliovirus with at least a part of the P1 of a Poliovirus (Sabin)selected from the groups consisting of PV1(S), PV2(S) and PV3(S);

[0082] c. optionally substituting at least a part of the P3 of thepoliovirus with at least a part of the P3 of Poliovirus (Sabin),selected from the groups consisting of PV1(S), PV2(S) and PV3(S);

[0083] d. optionally, substituting at least a part of the 3′NTR of thepoliovirus with at least a part of the 3′NTR of poliovirus (Sabin),selected from the group consisting of PV1(s), PV2(S), and PV3(S); and

[0084] B. Administering intravenously, intrathecally or directly to thetumor site a composition comprising the recombinant poliovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0085]FIG. 1 depicts the genomic organization of poliovirus. The viralRNA is covalently linked to a genome-linked protein, VPg.5′NTR domain Iis also known as the cloverleaf. The open reading frame is divided intocoding regions for the structural (capsid) proteins (P1) and thenon-structural proteins (P2 and P3). Individual 5′NTR domains areindicated by roman numerals.

[0086]FIG. 2 is a representation of the predicted secondary structure ofthe poliovirus IRES (sequence and nucleotide numbering of PV1(M)). Allpicornaviruses (including poliovirus and HRV) feature IRES elementswithin their respective 5′NTRs. Poliovirus IRESes like theircounterparts from the genus RhInovIrus are type 1 IRESes. Wimmmer, etal., supra. Domains are numbered with roman numerals. The 154 nt spacerseparating a conserved silent AUG triplet within the base stem loop VI(nt #583) from the initiating AUG (position #743) has been omitted.

[0087]FIGS. 3A and 3B demonstrate the results of neuropathogenicitytesting of PV1(RIPO) and PV1(RIPOS) in CD155 tg mice as well as inCynomolgous monkeys. FIG. 3A shows the results of intraspinalinoculation. FIG. 3B shows the result of the intravenous andintracerebral inoculations.

[0088]FIG. 4 presents one-step growth curves in SK-N-MC neuroblastomacells of IRES chimeras featuring the IRES elements of Human Rhinovirustype 2 and type 14 (HRV2 and 14). Coxsackievirus B4 (CB4) and Echovirus9 (E9) with poliovirus P1, P2 and P3 respectively. Growth properties inHeLa cells of all these recombinants were undistinguishable from thoseof wild-type poliovirus (data not shown).

[0089]FIG. 5 is a schematic representation of PV/HRV2 IRES chimeras(HRV2-specific sequences are boxed). All chimeras feature the cloverleaf(5′NTR domain I), open reading frame and 3′NTR of PV1(M). The right handcolumn provides neuropathogenic indices obtained by intracerebralinoculation of individual recombinants into CD155 tg mice.

[0090]FIGS. 6A and 6B show one-step growth curves of those PV/HRV2 IRESchimeras that were found to be of attenuated neurovirulence in CD155 tgmice in SK-N-MC cells (FIG. 6A) and HeLa cells (FIG. 6B). For geneticstructure see FIG. 5. For comparison, growth kinetics of theneuropathogenic PV1(M) are included. Note that the non-neuropathogenicphenotype in experimental animals of PV1(R2-4, 6), PV1(R5), PV1(R5-6),PV1(R2-5), and PV1(R6) in CD155 tg mice (FIG. 5) is also evident intissue culture.

[0091]FIG. 7 depicts the IRES sequence and bigeneric structure ofPV1(prr) carrying the IRES of PV1(M) where the terminal loop regions ofdomain V (nt #484-nt #508) and domain VI (nt #594-nt #612) have beensubstituted with the corresponding fragments of HRV2 (boxed sequencesare derived from HRV2, the remaining sequences are from PV1(M)). Arestriction site for endonuclease KpnI that was introduced for cloningpurposes is boxed. The initiating AUG triplet is shown in white letters.

[0092]FIGS. 8A and 8B show growth kinetics of PV1(prr) in SK-N-MCneuroblastoma (FIG. 8A) and HeLa (FIG. 8B) cells in comparison to thoseof PV1(M) and PV1(RIPO). FIG. 8C demonstrates the results of an analysisof neuropathogenicity of PV1(prr) in CD155 tg mice.

[0093]FIG. 9 are one-step growth curves of PV1(M) (open symbols) andPV1(RIPO) (solid symbols) in HTB-14 (circles) and HTB-15 (triangles)glioblastoma cell lines, and in SK-N-MC neuroblastoma (squares) cells.The efficient replication of PV1(RIPO) in glioblastoma cells is in sharpcontrast with the poor growth capacity in neuroblastoma cells.

[0094]FIG. 10 one-step growth curves of PV1(RIPO) in a panel ofdifferent glioblastoma cell lines.

[0095]FIG. 11 one-step growth curves of PV1(RIPO) in a medulluoblastomacell line.

[0096]FIG. 12 one-step growth curves of PV1(RIPO) in a mammary carcinomacell line.

[0097]FIG. 13 one-step growth curves of PV1(RIPO) in prostate carcinomacell lines.

[0098]FIG. 14 one-step growth curves of PV1(RIPO) in a colorectalcarcinoma cell line.

[0099]FIG. 15 one-step growth curves of PV1(RIPO) in hepatocellularcarcinoma cell lines.

[0100]FIG. 16 one-step growth curves of PV1(RIPO) in a bronchialcarcinoma cell line.

[0101]FIG. 17 one-step growth curves of PV1(RIPO) in epidermoidcarcinoma cell lines.

[0102] FIGS. 18A-18E are photomicrographs of histological sectionsthrough subcutaneously implanted glioblastomas (cell line HTB-15) inathymic mice. FIGS. 18A, 18C, and 18E show a tumor from an untreatedmice that had been growing for about 60 days. FIGS. 18B, 18D and 18F arebrain sections from a fellow mouse treated with a single intraneoplasticinoculation of PV1(RIPO) 30 days after tumor implantation show thedramatic results of therapy with oncolytic oliovirus recombinants.

[0103]FIG. 19 is a graphic representation of the replication kineticsand tumor necrosis induced by PV1(RIPO) after intraneoplasticinoculation into tumors derived from cell line HTB-15 implantedsubcutaneously into athymic mice. Athymic mice carrying subcutaneousgliomas received a single intravenous inoculation of 5×10⁷ PV1(RIPO) 30days after tumor implantation. The graph shows solid viral replicationwithin neoplastic tissue with rapid and drastic tumor shrinkage ascompared with the absence of virus propagation in liver (open circles)and brain (open squares). After 14 days the tumor was no longermacroscopically visible, precluding tumor isolation and determination ofweight.

[0104] FIGS. 20A-20D show the progression of neurological disease inathymic mice implanted with HTB-14 and harboring intracerebralglioblastomas (FIG. 20A) and the result of treatment with PV1(RIPO)administered via various routes. The graphs represent the progression ofclinically apparent neurological symptoms stemming from expandinghemispheric neoplasms. The ratio of surviving/affected animals and theaverage survival is indicated. FIG. 20A shows the results with notreatment. FIG. 20B shows the results for mice which were treatedintramuscularly with 5×10⁷ pfu PV1(RIPO) (FIG. 20B). FIG. 20C shows theresults of mice that were treated intravenously with 5×10⁷ pfu ofPV1(RIPO). FIG. 20D shows the results of intracerebral administration ofthe same amount of recombinant virus, demonstrating a cure.

[0105] FIGS. 21A-21D are brain sections of: normal control athymic mice(FIGS. 21A and 21B), untreated athymic mice harboring intraventricularimplanted gliomas, cell line HTb-14 (FIGS. 21C-21D) and athymic miceharboring intraventricular implanted gliomas that had received a singleintracerebral inoculation of PV1(RIPO) 12 days following tumorimplantation (FIG. 21E). FIG. 21E shows the dramatic reduction of tumormass. A tissue defect stemming from destruction of a paraventricularneoplastic lesion adherent to the lateral wall of the right ventricle isclearly visible (for details see FIG. 22).

[0106] FIGS. 22A-22C are detailed views of sections depicted in FIG. 21.FIG. 22A is a section through the lateral ventricle of a normal mouseshows the detached intact ependymal lining of the intact ventricle wall.FIG. 22B is a section of untreated athymic mice with glioma implantharbors globular neoplastic masses that are attached alongside theventricle walls. FIG. 22C shows that lesions like the one shown in FIG.22B are destroyed upon treatment of tumor-bearing athymic mice withPV1(RIPO). Only the remains of a vigorous host reaction against theinvading tumor leaving a paraventricular parenchymal lesion indicatingthe site where a tumor fragment had attached to the ventricular wall.

DETAILED DESCRIPTION OF THE INVENTION

[0107] According to the present invention non-neuropathogenic oncolyticpoliovirus chimeras have been bio-engineered for the treatment ofmalignant tumors in various organs. The non-neuropathogenic oncolyticpoliovirus chimeras comprise

[0108] A recombinant poliovirus constructed from a poliovirus having a5′NTR region containing an internal ribosomal entry site (IRES), and thecoding sequences for structural proteins (P1), and for thenon-structural proteins (P2 and P3) and a 3′NTR selected from the groupconsisting of wild type serotype 1, serotype 2, and serotype 3, wherein

[0109] a. i. a part of the IRES of the poliovirus is substituted with apart of the IRES of Human Rhinovirus serotype 2 also having a 5′NTRregion containing an internal ribosomal entry site (IRES), the codingsequences of structural proteins (P1), and for the non-structuralproteins (P2 and P3) and a 3′NTR, or

[0110] ii. at least a part of the IRES of the poliovirus is substitutedwith at least a part of the IRES of a virus selected from the group ofpicornaviruses comprising Human Rhinovirus serotype 1-100,coxsackievirus serotype B1-B6, human echovirus serotype 1-7, 9, 11-27,29-33, all of which also having a 5′NTR region containing an internalribosomal entry site (IRES), the coding sequences of structural proteins(P1), and for the non-structural proteins (P2 and P3) and a 3′NTR, andwherein

[0111] b. optionally, at least a part of the P1 of the poliovirus issubstituted with at least a part of the P1 of a Poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S) and PV3(S);

[0112] c. optionally, at least a part of the P3 of the poliovirus issubstituted with at least a part of the P3 of Poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S) and PV3(S); and

[0113] d. optionally, at least a part of the 3′NTR of the poliovirus issubstituted with at least a part of the 3′NTR of poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S), and PV3(S).

[0114] The invention is further directed to a therapeutic method oftreating malignant tumors comprising the steps:

[0115] A. Preparing a recombinant poliovirus constructed from apoliovirus having a 5′NTR region containing an internal ribosomal entrysite (IRES), and the coding sequences for structural proteins (P1), andfor the non-structural proteins (P2 and P3) and a 3′NTR selected fromthe group consisting of wild type serotype 1, serotype 2, and serotype3, by

[0116] a. substituting at least a part of the IRES of the polioviruswith at least a part of the IRES of a virus selected from the group ofpicornaviruses comprising Human Rhinovirus serotype 1-100,coxsackievirus serotype B1-B6, human echovirus serotype 1-7, 9, 11-27,29-33, all of which also having a 5′NTR region containing an internalribosomal entry site (IRES), the coding sequences of structural proteins(P1), and for the non-structural proteins (P2 and P3) and a 3′NTR;

[0117] b. optionally substituting at least a part of P1 of thepoliovirus with at least a part of the P1 of a Poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S) and PV3(S);

[0118] c. optionally substituting at least a part of the P3 of thepoliovirus with at least a part of the P3 of Poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S) and PV3(S);

[0119] d. optionally, substituting at least a part the 3′NTR of thepoliovirus with at least a part of the 3′NTR of poliovirus (Sabin),selected from the group consisting of PV1(s), PV2(S), and PV3(S); and

[0120] B. Administering directly to the tumor site or intravenously acomposition comprising the recombinant poliovirus.

[0121] Structure and Characteristics of Recombinant Polioviruses

[0122] A prototype non-pathogenic poliovirus chimera has been generatedby exchanging the native IRES of type 1 poliovirus (Mahoney) with itscounterpart from Human Rhinovirus type 2 (HRV2), yielding PV1(RIPO).Other IRES chimeras have been developed using the procedure which led tothe construction of PV1(RIPO). The exchange of the poliovirus IRES withany of the IRES elements derived from the group of viruses comprisingHuman Rhinovirus type 1, 3-100, Coxsackievirus B1-B6(CB), and Echovirustype 1-7, 9, 11-27 and 29-33 is expected to provide a recombinantpoliovirus chimeras with a reduced ability to replicate within cells ofneuronal origin to lyse them. This is demonstrated by the one-stepgrowth curves of IRES chimeras of PV1(M) with HRV14 (PV/HRV14), with CB4(PV/CB4), and with E9 (PV/E9) in SK-N-MC cells (FIG. 4). The reductionor elimination of non-neurocytopathogenic phenotype of poliovirus IRESchimeras was confirmed in studies using CD155 tg mice (data not shown).

[0123] Novel non-neuropathogenic, oncolytic, recombinant chimeras of PV,PV(S) and HRV2 have been further constructed. In the novel chimericpolioviruses only a portion of the IRES of a wild type poliovirus, suchas PV1(M), has been substituted with the corresponding portion of HRV2(FIG. 5). In order to identify the specific portions, which arereplaced, the known domains II, III, IV, V, and VI of the IRES have beenutilized. In PV1(R2-4,6) domains II, III, IV, and VI of PV1(M) werereplaced with the domains II, III, IV and VI of HRV2; in PV1(R5), thedomain V of PV1(M) was replaced with the domain V of HRV2; etc.Polioviruses that carry bi-generic IRESes composed of sequence elementsderived from the IRES domains V and VI of HRV2 and PV1, respectively(see FIG. 5), are characterized by a loss of neuropathogenic potentialwhen tested in CD155 tg mice (FIG. 5). This phenotype was also evidentwhen one-step growth curves of these chimeras were established inSK-N-MC neuroblastoma cells (FIG. 6A).

[0124] The poliovirus recombinants which are suitable for the presentinvention feature a loss of neuropathogenic potential and hence are safeto use in human therapy. Ablated neuropathogenicity was documented inCD155 tg mice and non-human primates (FIG. 3) and in SK-N-MCneuroblastoma cells In vItro (FIG. 6). The non-neuropathogenic oncolyticpoliovirus recombinants are those wherein the IRES of wild typepoliovirus has been replaced with the IRES of HRV2. The replacement maybe in whole as in PV1(RIPO), or the replacement may be in part, whereina portion of the IRES of the wild type poliovirus is replaced with thecorresponding portion of the IRES of HRV2. For example, suitablechimeras may be represented as PV1(R2-4,6), PV1(R5), PV1(R2-5),PV1(R5-6), PV1(R6) and PV1(prr). PV1(prr) is a poliovirus recombinantwherein nucleotides 484-508 (nt #484-nt #568) of domain V andnucleotides 594-612 (nt #594-nt #612) of domain VI of the IRES of wildtype poliovirus were replaced with their counterpart nt #484-nt #508 ofdomain V and nt #594-nt #612 of domain VI from HRV2. See FIG. 7.PV1(prr) was characterized by a loss of neuropathogenicity, demonstratedby its reduced ability to propagate within cells of neuronal origin andfailure to cause neurological disease in CD155 tg mice (FIG. 8). Thepreferred poliovirus chimeras for the purposes of the invention arePV1(RIPO) and PV1(RIPOS).

[0125] In addition to the IRES element, a part of or the entire codingregion for the structural proteins (P1), non-structural proteins (P3)and/or the 3′NTR of the wild type PV may be replaced with thecorresponding part of or the entire coding region for the structuralproteins (P1), non-structural proteins (P3) and/or the 3′NTR of anyvirus strain of the group comprising PV1(S), PV2(S) and PV3(S). It isknown that important genetic determinants for attenuation ofneurovirulence may reside within the coding regions for the capsidproteins (P1), the non-structural protein (P3) or the 3′NTR of the Sabinstrains of poliovirus. Inclusion of these genetic markers residingwithin the coding regions for P1, P3 or the 3′NTR into oncolyticnon-pathogenic polioviruses will further ensure the ablation ofneurovirulence of the poliovirus recombinants or chimeras of the presentinvention.

[0126] Synthesis of Recombinant Polioviruses

[0127] Recombinant poliovirus chimeras can be synthesized by well-knownrecombinant DNA techniques. Any standard manual on DNA technologyprovides detailed protocols to produce the poliovirus chimeras of theinvention. Sambrook, Fritsch and Maniatis, Molecular Cloning, ColdSpring Harbor Laboratory Press, NY (1989).

[0128] The construction of a prototype recombinant poliovirus PV1(RIPO)was described in Gromeier, M., et al., supra. The cloning proceduresused to produce oncolytic polioviruses with attenuated neurovirulence isgenerally as follows. Exemplary detailed cloning instructions for theconstruction of such recombinant viruses are provided in the Examples.

[0129] A cloning cassette, allowing for the convenient exchange ofheterologous recombinant IRES elements into the poliovirus genome, isobtained through the introduction of engineered endonuclease restrictionsites positioned at nt #110 (adjacent to the 5′ border of the IRESelement) and nt #747 (immediately downstream of the initiating AUGtriplet). The latter restriction site, positioned within the openreading frame, is created through the introduction of silent mutations(described in Gromeier, M. et al., supra). The resulting cloningcassette can be used to easily integrate IREs elements:

[0130] (1) derived in toto from other virus species;

[0131] (2) generated by combining RNA structural domains from IRESelements of different virus species;

[0132] (3) generated by combining sequence fragments or individualnucleotides from different virus species;

[0133] (4) derived from eukaryotic sequences with IRES function; and

[0134] (5) those that are entirely synthetic.

[0135] Experimental results show that composite IRES elementsconstructed from individual structural domains or subdomain fragmentsoriginating from different virus species can replace the poliovirus IRESand give rise to novel recombinant viruses with favorable properties forthe use as oncolytic agents.

[0136] These composite IRES elements are constructed through the use ofpolymerase chain reaction (PCR)-generated fragments. The fragments arethose with cohesive ends forming endonuclease restriction sites that areeither engineered or already present in the IRES sequence used.Sequences within IRES elements that allow for the introduction of novelendonuclease restriction sites through mutagenesis have been empiricallyidentified. A detailed description for the cloning of exemplarycomposite IRES elements combining RNA structural domains or subdomainsequence elements derived from divergent virus species is given in theExamples 1 and 6.

[0137] A cloning cassette, allowing for the convenient exchange of theP1 coding region for the structural proteins with its counterparts fromthe group comprising PV serotype 1 (Sabin) is obtained through theintroduction of an engineered endonuclease restriction site (byintroduction of silent mutations) positioned at nt #3278, within the 5′most part of P2 bordering the 3′ limit of P1. An engineered endonucleaserestriction site positioned at nt #747 that has already been introducedwith the purpose of convenient IRES exchange forms the 5′ border of P1.Thus, the resulting cloning cassette, in addition to provide easyreplacement of the IRES, can serve to integrate any desired P1 codingregion selected from the group of polioviruses including the wildserotypes 1, 2 and 3, as well as the Sabin serotypes 1, 2 and 3. Forthis purpose, the P1 coding region from the selected strain is PCRamplified, making use of the cohesive ends generated by engineeredendonuclease restriction sites defining the borders of P1 in the novelcloning cassette.

[0138] A cloning cassette, allowing for the convenient exchange of thecoding region for the RNA-dependent RNA polymerase 3D^(pol) ofpoliovirus with its counterpart from a virus selected from the groupcomprising PV serotype 1 (Sabin), serotype 2 (Sabin), and serotype 3(Sabin) in constructed as follows. Unique endonuclease restriction sitesare introduced in the 5′ most part of 3D^(pol) at nt #6060, upstream ofany mutations within this coding region specific for any of the PV(Sabin) strains and in the 3′ most part of 3D^(pol) at nt #7330,downstream of any mutations within this coding region specific for anyof the PV (Sabin) strains. Any desired sequence encoding 3D^(pol)produced by PCR amplification from the viral cDNA in question can beintegrated into the cloning cassette making use of the introducedartificial restriction endonuclease recognition bordering the codingregion for 3D^(pol).

[0139] A cloning cassette, allowing for the convenient exchange of the3′NTR of poliovirus with the 3′NTR of the group comprising PV1(Sabin),PV2(Sabin), and PV3(Sabin) is constructed as follows. An engineeredunique restriction site within the 3′ most region of 3D^(pol), at nt#7330, has already been introduced for the creation of a cloningcassette for the convenient exchange of the coding region for 3D^(pol).An additional restriction site is introduced at the very 3′ border ofthe viral genome, immediately preceding the poly(A) tail from the 3′restriction site of the cloning cassette for easy exchange of the 3′NTR(nt #7439). PCR amplification of the desired 3′NTR from any given viralstrain can easily be inserted into the cloning cassette making use ofthe engineered restriction sites defining in the 3′ most part of P3 andimmediately preceding poly(A).

[0140] Combining the genome modifications described above is obtained, apoliovirus cDNA with 4 independent cloning cassettes allowing for simpleexchange of:

[0141] (1) IRES elements

[0142] (2) the coding region for the structural proteins P1

[0143] (3) the coding region for 3D^(pol)

[0144] (4) the 3′NTR

[0145] This “multipurpose cloning cassette” may be used to obtainrecombinant polioviruses of the invention, including any poliovirusselected from the group of viruses comprising serotype 1, serotype 2 andserotype 3, wherein,

[0146] a. at least a part of the IRES is substituted with at least apart of the IRES of a virus selected from the group of picornavirusescomprising Human Rhinovirus serotype 1-100, coxsackievirus serotypeB1-B6, human echovirus serotype 1-7, 9, 11-27, 29-33, also having a5′NTR region containing an internal ribosomal entry site (IRES),

[0147] b. optionally, at least a part of P1 is substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S);

[0148] c. optionally, at least a part of P1 is substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S); and

[0149] d. optionally, at least a part of the 3′NTR is substituted withthe corresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S).

[0150] Experiments are presented herebelow describing virus recombinantscarrying composite IRES elements composed out of domains II, III, IV, V,VI derived from divergent virus species. The general cloning proceduresfor exemplifying intergeneric IRES domain recombinants (displayed inFIG. 5) are as follows (for detailed instructions, refer to Example 1).

[0151] Synthetic IRES elements that contain RNA structural domainsderived from divergent virus species can be constructed if structuralintegrity essential for efficient IRES function is maintained. A seriesof intergeneric IRES domain and subdomain recombinants that combine IRESsequence elements of polio and HRV2 have been developed. Theserecombinants IRESes can be produced through PCR amplification of desiredIRES fragments using introduced endonuclease restriction sites for theformation of cohesive ends needed for cloning purposes as follows. PCRamplification using primers that carry recognition sites forendonucleases can produce individual IRES stem loops, or subdomain IRESfragments carrying cohesive ends for ligation into intact IRES units andsubsequent integration into virus cDNA cloning cassettes. The positionof mutations introduced for the creation of restriction endonucleaserecognition sites has to be determined empirically, because they mayinterfere with IRES function. Suitable restriction sites that do notinterfere with IRES function for the intergeneric IRES domainrecombinants are provided in Example 6.

[0152] Similarly, additional modifications of IRES elements through theintroduction of artificial endonuclease restriction sites may beintroduced for the synthesis of novel intergeneric IRES chimeras thatrecombine sequence elements of different viruses in alternative ways. Inaddition to intergeneric domain recombinants, artificial IRES elementscan be generated through the exchange of subdomain IRES fragments withtheir corresponding regions originating from a different virus species.Subdomain fragment chimeras that feature IRES elements in which only fewnucleotides have been exchanged with the corresponding residues of adifferent virus species are described in Examples 6.

[0153] In principal, experimental procedures required to producesubdomain IRES chimeras are identical to those employed for thegeneration of domain IRES chimeras described above. PCR fragmentsgenerated from IRES element of the desired species origin are generatedmaking use of cohesive ends created through the introduction ofartificial endonuclease restriction sites following the parameters formaintenance of IRES function. Subsequently, IRES subdomain fragmentchimeras can be produced through the ligation of different PCR productsharboring engineered nucleotide exchanges with cohesive ends asdescribed above.

[0154] The resulting intradomain hybrid IRES elements can be integratedinto any poliovirus cDNA cloning cassette. Any IRES element, intactheterologous IRESes, domain chimeric IRESes, subdomain chimeric IRESes,or entirely non-viral or synthetic IRES elements can be integrated intothe poliovirus cDNA cassette with great ease. For that purpose thecloning cassette is digested with the endonucleases flanking the IRESintegration sites (nt #110, and nt #747) and the desired IRES elementsfeaturing cohesive ends corresponding to those generated by endonucleasedigestion of the cloning cassette is ligated into the cDNA.

[0155] Following these general instructions poliovirus recombinants canbe generated using intact heterologous IRES elements, domain chimericIRESes, or subdomain chimeric IRESes of a virus selected from the groupof picornaviruses comprising poliovirus serotype 1-3, polioviruses(Sabin) serotype 1-3, Human Rhinovirus serotype 1-100, coxsackievirusserotype B1-B6, human echovirus serotype 1-7, 9, 11-27, 29-33, allhaving a 5′NTR region containing, an internal ribosomal entry site(IRES). Composite IRES elements can be integrated into a poliovirusselected from the group comprising PV serotype 1, serotype 2, andserotype 3, containing the coding sequences for structural proteins(P1), and for the non-structural proteins (P2 and P3) and a 3′NTRselected from the group consisting of wild type serotype 1, serotype 2,and serotype 3, wherein

[0156] a. optionally, at least a part of P1 is substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S)

[0157] b. optionally, at least a part of P3 is substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S)

[0158] c. optionally, at least a part of the 3′NTR is substituted withthe corresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S).

[0159] Alternatively, the recombinant poliovirus maybe synthesized invitro in accordance with the procedure described in Wimmer, et al., U.S.Pat. No. 5,674,729, incorporated herein by reference. The procedure isgenerally as follows.

[0160] Preparing a lysate from mammalian cells such as kidney cells,epithelial cells, liver cells, cells of the central nervous system,fibroblastic cells, transformed or tumorigenic cell lines thereofincluding HeLa cells, hepatoma cells and L cells; wherein the nuclei andmitochondria were removed; and the endogenous mRNA deactivated withmicrococcal nuclease, calcium chloride and EGTA. The preparing of an invitro synthesis medium by mixing: the lysate prepared above with thefollowing materials to arrive at a final concentration in the mixture ofabout 1 mM ATP, about 20 μM to 1000 μM each of GTP, CTP and UTP, about10 mM creatine phosphate, about 24 μg/mL creatine phosphokinase, about 2mM dithiothreitol, about 24 μg/mL calf liver t-RNA, about 12 μM each of20 amino acids, about 18 mM Herpes, pH 7.4, about 240 μM spermidine,about 50 mM to 200 mM potassium acetate, and about 1 mM to 4 mM ofMgCl₂. Then adding isolated viral RNA from virus or in vitro synthesizedviral RNA prepared from cDNA to the in vitro synthesis medium; andincubating the viral RNA for about 2 to 24 hrs. at a temperature fromabout 30° C. to 40° C.

[0161] Determination of Neuropathogenicity

[0162] The neuropathogenicity of poliovirus chimeras may be determinedby following the standardized protocols of testing PV Sabin strains(oral PV vaccines). Generally, neurovirulence is determined in CD155-tgmice and Cynomolgus monkeys. CD155-tg mice were infected by either theintravenous (i.v.) or the intracerebral (i.c.) route and the clinicalcourse of the ensuing neurological disease was monitored. Animal centralnervous tissues were analyzed histopathologically and assayed for viralreplication. Cynomolgus monkeys were inoculated intraspinally with 10⁶CCID₅₀/mL (50% cell culture infectious doses/mL). Monkeys weresacrificed 17 days after intraspinal inoculation and the extent anddistribution of spinal histopathology was assessed in a manner describedby Omata, et al., J. Virol., 58:348-358 (1986). Lesion scores weredetermined by established procedures. WHO Technical Report Series No. 80(1990); Kawamura, N., et al., J Virol., 63:1302-1309 (1989).

[0163] Assessment of the Oncolytic Properties

[0164] Oncolytic properties of the poliovirus chimeras of the inventionwere assessed by the in vitro growth of the chimeric viruses in a panelof cell lines derived from human malignancies. The procedure isdescribed herebelow.

[0165] Cell lines originally obtained from surgical excised tumors andpropagated in tissue culture are tested for susceptibility to oncolyticpolioviruses in one-step growth curves as follows. Monolayer cellcultures (ca. 5×10⁶ cells per plate) of the line in question are grownand infected at a multiplicity of infection (MOI) of 10. Infected cellsare gently shaken for 30 min. at room temperature to allow for virusbinding. Subsequently, cell monolayers are rinsed 5 times with 5 ml ofserum-free medium each to remove unbound virus. Finally monolayers areoverlaid with 2 ml of growth medium containing 2% of fetal calf serumand placed at 37° C. At defined time points (0, 2, 4, 6, 8, 10, 12, 24hrs.) post infection (p.i.) cell culture dishes are frozen to stop theinfectious process. At the completion of the experiment all collectedsamples are subjected to 4 consecutive freeze/thaw cycles to break openinfected cells. The material thus treated is then analyzed with a plaqueassay to determine the total amount of infectious virus present at eachtime point p.i. To this end serial dilutions of each sample are producedand used to infect HeLa cell monolayers that are overlaid with 3% Nobleagar containing growth medium. The amount of infectious virus can bedetermined by counting the plaques of infected and lysed cells thatformed underneath the solidified agar corresponding to the number ofinfectious particles present within the sample. The quantity ofinfectious particles at various time points is plotted against time postinfection (p.i.). The growth curve thus obtained represents an accuratereflection of the replication and hence oncolytic capacity of the virusstrain tested in that particular cell line.

[0166] The oncolytic properties of the poliovirus chimeras of thepresent invention may also be assessed in vivo as follows. Experimentaltumors are produced in athymic mice by subcutaneous or stereotacticintracerebral implantation of malignant cells. Tumor progression inuntreated athymic mice and athymic mice that have been administeredoncolytic poliovirus recombinants following various treatment regimensare followed by clinical observation and pathological examination. Thetechnique of tumor implantation into athymic mice is standard proceduredescribed in detail in Fogh, J., et al., J. Natl. Cancer Inst.,59:221-226 (1977).

[0167] Pharmaceutical Compositions and Treatment Methods

[0168] The poliovirus chimeras of this invention are useful inprophylactic and therapeutic compositions for treating malignant tumorsin various organs, such as: breast, colon, bronchial passage, epitheliallining of the gastrointestinal, upper respiratory and genito-urinarytracts, liver, prostate and the brain.

[0169] The most preferred pharmaceutical compositions of this inventionfor adminstration to humans comprise the poliovirus chimeras, PV1(RIPO)and PV1(RIPOS).

[0170] The pharmaceutical compositions of this invention may furthercomprise other therapeutics for the prophylaxis of malignant tumors. Forexample, the poliovirus chimeras of this invention may be used incombination with surgery, radiation therapy and/or chemotherapy.Furthermore, one or more poliovirus chimeras may be used in combinationwith two or more of the foregoing therapeutic procedures. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities oradverse effects associated with the various monotherapies.

[0171] The pharmaceutical compositions of this invention comprise atherapeutically effective amount of one or more poliovirus chimerasaccording to this invention, and a pharmaceutically acceptable carrier.By “therapeutically effective amount” is meant an amount capable ofcausing lysis of the cancer cells to cause tumor necrosis. By“pharmaceutically acceptable carrier” is meant a carrier that does notcause an allergic reaction or other untoward effect in patients to whomit is administered.

[0172] Suitable pharmaceutically acceptable carriers include, forexample, one or more of water, saline, phosphate buffered saline,dextrose, glycerol, ethanol and the like, as well as combinationsthereof. Pharmaceutically acceptable carriers may further comprise minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the poliovirus chimeras.

[0173] The compositions of this invention may be in a variety of forms.These include, for example, liquid dosage forms, such as liquidsolutions, dispersions or suspensions, injectable and infusiblesolutions. The preferred form depends on the intended mode ofadministration and prophylactic or therapeutic application. Thepreferred compositions are in the form of injectable or infusiblesolutions.

[0174] Therapeutic oncolytic polioviruses can be deliveredintravenously, intrathecally or intraneoplastically (directly into theprimary tumor). The preferred mode of administration is directly to thetumor site. For all forms of delivery, the recombinant virus is mostpreferably formulated in a physiological salt solution: e.g. HANKSbalanced salt solution [composition: 1.3 mM CaCl₂ (anhyd.), 5.0 mM KCl,0.3 mM KH₂PO₄, 0.5 mM MgCl₂. 6H2O, 0.4 mM MgSO₄.7H₂O, 138 mM NaCl, 4.0mM NaHCO₃, 0.3 mM Na₂HPO₄, 5.6 mM D-Glucose]. The inoculum of virusapplied for therapeutic purposes can be administered in an exceedinglysmall volume ranging between 1-10 μl. Recombinant polioviruses stored ina physiological salt solution of the composition detailed above can bestored at −80° C. for many years with minimal loss of activity. Shortterm storage should be at 4° C. At this temperature virus solutions canbe stored for at least one year with minimal loss of activity.

[0175] It will be apparent to those of skill in the art that thetherapeutically effective amount of poliovirus chimeras of thisinvention will depend upon the administration schedule, the unit dose ofpoliovirus chimeras administered, whether the poliovirus chimera isadministered in combination with other therapeutic agents, the statusand health of the patient.

[0176] The therapeutically effective amounts of oncolytic recombinantvirus can be determined empirically and depend on the maximal amount ofthe recombinant virus that can be administered safely, and the minimalamount of the recombinant virus that produces efficient oncolysis.Experiments studying the effect of intraspinal inoculation of candidateoncolytic polioviruses into non-human primates (FIG. 3) indicate that adose of 5×10⁶ pfu of PV1(RIPO) or PV1(RIPOS) can be used forintracerebral, intraspinal or intrathecal administration without thedanger of inducing any neurological sequelae. Based on the Cynomolgusmonkey data, weighing just about 7.0 pounds, the appropriate dose for anaverage human (e.g. 140 pounds) is about 1×10⁸ pfu of virus. Maximalvirus delivery appeared to be beneficial to achieve maximal oncolysis inanimal experiments. Thus, the virus inoculums used for intraneoplasticinjections into humans would be in the range of 1×10⁶ to 5×10⁸ pfu.However, the dose may be adjusted in accordance with the particularrecombinant poliovirus contemplated and the route of administrationdesired.

[0177] Intraneoplastic inoculations of oncolytic polioviruses producedsignificantly better oncolysis rates than intravenous administration inexperimental animals (FIG. 20). Based on the data obtained, therecombinant polioviruses of the present invention are non-neurovirulentand non-pathogenic. The mechanism by which oncolysis takes place is bythe ability of these recombinant polioviruses to replicate in the cancercells at a rate which causes the cells to “explode”. The recombinantpolioviruses of the present invention do not affect normal cellularprocesses and are thus not expected to be toxic to normal cells.Therefore, it would appear that there is no upper limit to the doselevel which can be administered. Thus, to produce the same oncolyticeffect achieved through intraneoplastic inoculation of virus by theintravenous route, significantly higher amounts of virus should be andcould be administered. However, in an abundance of caution, theappropriate dose level should be the minimum amount which would achievethe oncolytic effect.

[0178] Therapeutic inoculations of oncolytic polioviruses can be givenrepeatedly, depending upon the effect of the initial treatment regimen.Since poliovirus exists in three antigenically distinct serotypes,candidate oncolytic polioviruses, e.g. PV1(RIPO), will be available asthree different serotypes, e.g. PV1(RIPO), PV2(RIPO), PV3(RIPO). Shouldthe host's immune response to a particular oncolytic poliovirusadministered initially limit its effectiveness, additional injections ofan oncolytic poliovirus with a different poliovirus serotype can bemade. The host's immune response to a particular poliovirus can beeasily determined serologically. It will be recognized, however, thatlower or higher dosages than those indicated above according to theadministration schedules selected.

[0179] For that purpose, serological data on the status of immunityagainst any given poliovirus can be used to make an informed decision onwhich variant of the oncolytic polioviruses to be used. For example, ifa high titer against poliovirus serotype 1 is evident throughserological analysis of a candidate patient for treatment with oncolyticnon-pathogenic polioviruses, a serotype 2 or −3 variant of thetherapeutic virus preparation should be used for tumor therapy.

[0180] In order that this invention may be better understood, thefollowing examples are set forth. These examples are for purposes ofillustration only, and are not to be construed as limiting the scope ofthe invention.

EXAMPLE 1 Construction of Intergeneric PV Recombinants

[0181] Construction of PV1(RIPO)/3DS/3'S

[0182] The construction of a prototype recombinant poliovirus PV1(RIPOS)was described in Gromeier, M., et al., Proc. Natl. Acad. Sci. USA.91:1406-1410 (1996), incorporated herein by reference.

[0183] PV1(RIPOS/3DS/3'S), having a genome of PV1(M) with a 5′NTR regioncontaining an IRES derived from HRV2, the coding region for thestructural proteins P1 derived from PV1(S), the coding region for theviral RNA-dependent RNA polymerase 3D^(pol) (a part of P3) and the 3′NTRderived from PV1(S) was constructed as follows.

[0184] A plasmid containing the cDNA of PV1(M) with an engineeredrestriction site for endonuclease EcoRI at nt #110 was used to produce acloning cassette suitable for simple exchange of IRES segments. Theplasmid used was obtained from R. Andino (UCSF) and is labeled pPN6. Afragment encompassing the HRV2 IRES flanked by restriction sites forEcoRI and SacI was generated by PCR using a HRV2 cDNA (obtained fromKuechler, D., University of Vienna, Austria) as template with primers5′CCGAATTCAACTTAGAAGTTTTTCACAAAG-3′ (SEQ ID NO:1) and5′-CCTGAGCTCCCATGGTGCCAATATATATATTG-3′ (SEQ ID NO:2).

[0185] In similar manner the IRES elements of HRV14, Coxsackievirus B4(CBV4), and Echovirus 9 (E9) where inserted into the poliovirus IREScloning cassette. For that purpose, the IRES was PCR amplified fromHRV14 cDNA (kindly provided by E. Kuechler, University of Vienna,Austria) using primers 5′-CCGGAATTCCCACCCATGAAACGTTAG-3′ (SEQ ID NO:3)and 5′-CCTGAGCTCCATGATCACAGTATATG-3′ (SEQ ID NO:4); from CBV4 cDNA usingprimers 5′-CTTAGAATTCAAAGAAACAATGGTCAATTACTGACG-3′ (SEQ ID NO:5) and5′-CCTGAGCTCCCATTTTATCG-3′ (SEQ ID NO:6); and from E9 cDNA using primers5′-CCGAATTCAGAAGCATGACTCCAACGG-3′ (SEQ ID NO:7) and5′-GGGAGCTCCCATTTTGATGTATTGAGTGTTAA-3′ (SEQ ID NO:8). All PCR-generatedIRES elements from different piconaviruspecies featured EcoRI and SacIrestriction sites a their 5′ and 3′ ends, respectively, and could thusbe cloned into the poliovirus IRES cloning cassette as described forHRV2 above. A PCR-fragment encompassing a segment of the open readingframe encoding the viral structural proteins P1 [spanning a segmentimmediately upstream of the initiating AUG to a unique NheI restrictionsite at position #2978 within the PV1(M) genome] was generated usingprimers 5′-CCGAGCTCAGGTTTCATCACAG-3′ (SEQ ID NO:9) and5′-CCTGTGCTAGCGCTTTTTGCTC-3′ (SEQ ID NO:10) and pPN6 as a template. Theformer PCR product was digested with EcoRI and SacI, the latter withSacI and NheI and both fragments were ligated to pPN6 previously cutwith endonucleases EcoRI and NheI and treated with calf intestinalphosphatase. The resulting ligation product is PV1(RIPO), containing theIRES region of HRV2 within the genome of PV1.

[0186] Construction of PV1(RIPOS)

[0187] A fragment encompassing the region of P1 containing all aminoacid exchanges specifying the coding region for the structural proteinsof PV1(S) was generated by PCP using primers (SEQ ID NO:9) and (SEQ IDNO:10) and PV1(S) cDNA as a template. The resulting PCR product wasdigested with SacI and NheI and ligated to PV1(RIPO) treated previouslywith the identical endonucleases and calf intestinal phosphatase. Theresulting viral cDNA was that of PV1(RIPOS).

[0188] In order to insert the coding region of 3D^(pol) (a part of P3),the plasmid containing the cDNA of PV1(RIPOS) was cut with therestriction endonucleases BglII (nt #5600 in P2) and FspI (within thevector). The cloning strategy of PV1 (RIPOS/3Ds/3'S) followed theconstruction of a cloning cassette containing insertion sites framed byunique restriction sites for the rapid exchange of the coding region for3D^(pol) (restriction sites XhoI and BspEI) and the 3′NTR (restrictionsites BspEI and FspI). According to this strategy PCR was performedusing PV1(M) cDNA as template with primers5′-GGAGATCTTGGATGCCAAAGCGCTCGAAG-3′ (SEQ ID NO:11) and5′-GGCTCGAGCTTGGTTTTGGACGGGG-3′ (SEQ ID NO:12) generating a DNA fragmentencompassing parts of P3 (nt #5600-nt #6064), flanked by restrictionendonuclease recognition sites BglII and XhoI creating a novel XhoIrestriction site within the 5′ part of the coding region for 3D^(pol).An additional PCR reaction using primers5′-GGCTCGAGCCCAGTGCTTTCCACTATGTGTTTGAAGGGG-3′ (SEQ ID NO:13) and5′-TCCGGAAGCAATAAAGCTCTTCCAATTGG-3′ (SEQ ID NO:14) from PV1(S) cDNA as atemplate generated the coding region for 3D^(pol) of PV1(S) flanked byrestriction sites XhoI and BspEI, creating a novel BspEI site throughthe introduction of silent mutations with the 3′ part of the codingregion for 3D^(pol). A third PCR reaction from PV1(S) cDNA as a templateusing primers5′-GTCCGGAGTACTCAACATTGTACCGCCGTTGGCTTGACTCATTTTAGTAACCC-3′ (SEQ IDNO:15) and 5′-GGTGCGAACGTTGTTGCCATTGCTGC-3′ (SEQ ID NO:16) generated bythe 3′ NTR region of PV1(S) with a cohesive 5′ end through theintroduction of silent mutations within the 3′ end of the coding regionfor 3D^(pol) creating a restriction site for BspEI and a cohesive 3′vectorial fragment framed by the recognition site for restrictionendonuclease FspI. Ligation of all three PCR fragments into PV1(RIPOS)previously cut with BglII and FspI yielded PV1(RIPOS/3DS/3'S) with thedesired genotype: 5′cloverleaf [PV1(M)]-IRES [HRV2]-P1[PV1(S)]-P2/P3(excl. 3D^(pol)) [PV1(M)]-3D^(pol) [PV1(S)]-3′NTR[PV1(S)].

[0189] The procedure described above can be adapted to produce otherrecombinant polioviruses of the invention, including any poliovirusselected from the group of viruses comprising serotype 1, serotype 2,and serotype 3, wherein

[0190] (a) at least a part of the IRES is substituted with a part or allof the IRES of a virus selected from the group of picornavirusescomprising Human Rhinovirus serotype 1-100, coxsackievirus serotypeB1-B6, human echovirus serotype 1-7, 9, 11-27, 29-33, also having a5′NTR region containing an internal ribosomal entry site (IRES),

[0191] (b) optionally, at least a part of P1 is substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S)

[0192] (c) optionally, at least a part of P3 substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S)

[0193] (d) optionally, at least a part of the 3′NTR substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S).

[0194] Furthermore, experiments involving virus recombinants carryingcomposite IRES elements composed out of domains II, III, IV, V, and VIderived from divergent virus speciesa re described. The cloningprocedures for a number of exemplifying intergeneric IRES domainrecombinants (displayed in FIG. 5) are as follows.

[0195] Construct PV1(R2-4) was generated by ligating a PCR productencompassing the HRV2 IRES domains II-IV using primers (SEQ ID NO:1) and5′-CCGGATCCAAAGCGAGCACACGGGGC-3′ (SEQ ID NO:17) to a PCR productencompassing PV1(M) domains V and VI produced with primers5′-CCGGATCCTCCGGCCCCTGAATGCG-3′ (SEQ ID NO:18) and5′-CCTGAGCTCCCATTATGATACAATTGTCTG-3′ (SEQ ID NO:19).

[0196] For construct PV1(R2-4,6), primers (SEQ ID NO:1) and (SEQ IDNO:17) were used to generate a PCR fragment encompassing domains II-IVof the HRV2 IRES which was ligated to a PCR fragment obtained fromPV1(M) using primers (SEQ ID NO:18) and5′-GGTACCAATAAAATAAAAGGAAACACGGACACC-3′ (SEQ ID NO:20) corresponding toPV1(M) domain V and to the PCR product from HRV2 yielding domain VI withthe use of primers 5′-GCGGTACCGCTTATGGTGACAATATATAC-3′ (SEQ ID NO:21)and (SEQ ID NO:2).

[0197] For construct PV1(R2-5) primers (SEQ ID NO:1) and5′-CCGGTACCTAAAGGAAAAAGTGAAACA-3′ (SEQ ID NO:22) were used to generate afragment containing domains II-V of HRV2 that was ligated to domain VIof PV1(M), PCR synthesized with the use of primers5′-CCGGTACCGCTTATGGTGACAATCACAG-3′ (SEQ ID NO:23) and (SEQ ID NO:19).Construct PV1(R5-6) was generated by ligating a PCR product from PV1(M)using primers 5′-GGGAATTCAGACGCACAAAACCAAG-3′ (SEQ ID NO:24) and5′-CCGGATCCTTATGTAGCTCAATAGG-3′ (SEQ ID NO:25) with a PCR productencompassing domains V and VI from HRV2 generated with primers5′-CCGGATCCTCCGGCCCCTGAATGTGG-3′ (SEQ ID NO:26) and (SEQ ID NO:2).

[0198] Construct PV1(R5) was generated ligating a PCR product spanningPV1(M) domains II-IV produced with primers (SEQ ID NO:23) and (SEQ IDNO:24) to a PCR product encompassing domain V of HRV2 generated withprimers (SEQ ID NO:26) and (SEQ ID NO:22) and a PCR product representingdomain VI of PV1(M) produced with primers (SEQ ID NO:23) and (SEQ IDNO:19). PV1(R6) was the result of ligating a PCR product from a reactionusing primers (SEQ ID NO:24) and (SEQ ID NO:20) corresponding to IRESdomain II-V of PV1(M) to a PCR product generated with the use of primers(SEQ ID NO:21) and (SEQ ID NO:2) corresponding to HRV2 IRES domain VI.

[0199] Recombinant IRES elements combining IRES domains from differentpicornavirus species can be generated using fragments of the IRESelements of a virus selected from the group of picornaviruses comprisingpoliovirus serotype 1-3, polioviruses (Sabin) serotype 1-3, HumanRhinovirus serotype 1-1 00, coxsackievirus serotype B1-B6, humanechovirus serotype 1-7, 9, 11-27, 29-33, all having a 5′NTR regioncontaining an internal ribosomal entry site (IRES).

[0200] Composite IRES elements can be integrated into a poliovirusselected from the group comprising PV serotype 1, serotype 2, andserotype 3, containing the coding sequences for structural proteins(P1), and for the non-structural proteins (P2 and P3) and a 3′NTRselected from the group consisting of wild type serotype 1, serotype 2,and serotype 3.

[0201] Optionally, at least a part of P1 is substituted with thecorresponding region of one of the viruses selected from the groupconsisting of PV1(S), PV2(S), and PV3(S); or at least a part of P3 issubstituted with the corresponding region of one of the viruses selectedfrom the group consisting of PV1(S), PV2(S), and PV3(S); or, at least apart of the 3′NTR is substituted with the corresponding region of one ofthe viruses selected from the group consisting of PV1(S), PV2(S), andPV3(S).

[0202] Plasmids containing the cDNA of the resulting recombinant virusof the above mentioned genotype or any other variant were amplified,purified and digested with the restriction endonuclease FspI forlinearization (this endonuclease cuts within vectorial sequences). Theresulting linearized cDNA (which contains a recognition motif for theDNA-dependent RNA polymerase T7 preceding the 5′ insertion site of thevirus cDNA) was used for in vitro transcription using T7 polymerase toproduce full-length viral RNA. Viral RNA thus generated was used totransfect HeLa cells by the Dextran-sulfate method in order to produceinfectious virus. Transfected cells were observed for the occurrence ofthe cytopathic effect indicating productive poliovirus infection andinfectious virus will be propagated in HeLa cells, purified and frozenfor indefinite storage.

EXAMPLE 2 In Vitro Growth of PV Recombinants in Cultured Cells toDetermine Neurovirulence

[0203] Neurovirulence is tested in vitro and in vivo. For in vitrotesting, cell lines HEp-2, derived from a human laryngeal epidermoidcarcinoma, and SK-N-MC, derived from a neuroblastoma in a human subject,were obtained from ATCC and grown in Dulbecco's minimal essential medium(DMEM; GIBCO). 10% fetal bovine serum (GIBCO), penicillin (100 units/mL)and streptomycin (100 μg/mL). HEp-2 and SK-N-MC, and monolayers in 6 cm.plastic culture dishes were inoculated with a suspension of PV1(RIPO) orPV1(RIPOS) at a multiplicity of infection of 10 and gently shaken for 30min. at room temperature. Afterwards, the dishes were washed five timeseach with 5 mL of DMEM. Then the monolayers were overlaid with 2 mL ofDMEM containing 2% fetal bovine serum. Synchronized infection wasinterrupted at the indicated intervals, cell monolayers were lysed byfour consecutive freeze-thaw cycles, and the viral yield in the celllysate was determined in a plaque assay.

[0204] The attenuated phenotype of poliovirus has been documented to bereproducible in tissue culture. Agol, V. I., et al., J. Virol.,63:4034-4038 (1989). La Monica, N. & Racaniello, V. R., J. Virol.,63:2357-2360 (1989). Growth defects of attenuated strains of poliovirusevident in SK-N-MC neuroblastoma cell lines correlated with thedeficiency to cause poliomyelitic disease in Cynomolgus monkeys or CD155tg mice. Thus, the non-neuropathogenic phenotype, a prerequisite for theengineering of safe oncolytic polioviruses devoid of unwanted pathogenicproperties, can be ascertained with great ease and accuracy byestablishing one-step growth curves in SK-N-MC neuroblastoma cells asdescribed above.

[0205] The results are presented in FIGS. 3-6 and show thatneurovirulence or neuropathogenicity has been ablated in PV1(RIPO) andPV1(RIPOS). The non-neuropathogenic phenotype has been demonstrated fora great number of different recombinant IRES constructs described inthis application. These include polioviruses whose IRES elements havebeen entirely (FIG. 4) or in part (FIGS. 6, 8) substituted with thecorresponding entire IRES elements or partial IRES fragments derivedfrom various rhinoviruses (HRV2, HRV14), Coxsackie B virus (CBV4), andEchovirus (E9).

EXAMPLE 3 Determination of Neurovirulence in CD155-tg Mice andCynomolgus Monkeys

[0206] All PV strains containing either the homologous or theheterologous IRES elements were assayed to determine their neurovirulentpotential in mice transgenic for the human PV receptor, CD155-tg micestrain ICR.PVR.tgI, Koike et al., supra. Wild type (wt) PV strainsinduce in these animals a neurological disease indistinguishable,clinically and histologically, from primate poliomyelitis. CD155-tg micewere infected either by the i.c. or i.v. route and the clinical courseof ensuing neurological disease was monitored. Animal central nervoussystem tissues were analyzed histopathologically and assayed for viralreplication.

[0207] Groups of four mice were infected with a given amount of virusranging from 10² to 10⁴ pfu i.c. and 10³ to 10⁵ pfu i.v. for PV1(M) andPV1(R2-4); 10⁵ to 10⁸ i.v. and 10⁷ to 10⁹ i.c. for PV1(S), PV1(RIPO),PV1(RIPOS), PV1(R2-5), PV1(R6), PV1(R2-4,6), PV1(R5). CD155 tg mice thatwere inoculated with the various constructs were clinically observed andfatalities were recorded. LD₅₀ values were calculated by the method ofReed and Muench, Am. J. Hyg., 27:493-495 (1938).

[0208] Remarkably, PV1(RIPO) and PV1(RIPOS) were devoid of anyneurovirulent potential and produced only a transient subtle paraparesisin CD155 tg mice. Intracerebral injections with PV1(RIPO) or PV1(RIPOS)up to 10⁹ pfu did not cause any apparent alterations within the centralnervous system, and intraspinal replication of these recombinants wasabsent.

EXAMPLE 4 Determination of Oncolysis with PV Chimeras

[0209] PV1(RIPO) was constructed by exchanging the cognate PV IRES withthat of HRV2 (See FIG. 5). PV1(RIPO) is exceptional because of its lossof pathogenicity in CD155-tg mice, which develop poliomyelitis uponpoliovirus infection, Ren, et al., supra; Koike et al., supra; Gromeieret al., supra) and in non-human primates (Cynomolgus monkeys) afterintraspinal inoculation (FIG. 3).

[0210] Loss of neuropathogenicity of PV1(RIPO) was also evident by poorpropagation in cultured cell lines of neuronal derivation, a phenomenonthat correlates with cell-internal restriction of replication (FIG. 4).In contrast, propagation of PV1(RIPO) in rapidly dividing malignantcells, such as HTB-14 or HTB-15 glioblastoma cells (FIG. 9) or in HeLacells (FIG. 8) was near wild type levels. The locus of attenuation ofPV1(RIPO) was mapped to two stem-loop domains within the HRV2 IRES. SeeFIG. 5, which depicts PV variants with PV/HRV2 chimeric IRESes whereHRV2 IRES components are boxed. The combined IRES domains V/VI of PVwere required for the neuropathogenic phenotype, because only wild typePV1(M) and chimera PV1(R2-4) caused poliomyelitis in CD155-tg mice. Seeright column which indicates neuropathogenicity indices. PV1(RIPOS) thatcarries the P1 capsid encoding region of the live attenuated vaccinestrain PV1(Sabin) have also been constructed.

[0211] The oncolytic potential of recombinant polioviruses was assessedin tissue culture by the establishment of one-step growth curves intissue culture cell lines derived from excised human tumors. To thatend, cultured cells monolayer were grown and infected with the oncolyticpoliovirus recombinant in question. Infection was interrupted atpredetermined intervals and a one-step growth curve was establishedfollowing the procedures outlined on pg. 32 et seq.

[0212] Oncolytic potential of PV1(RIPO) has been determined by analysisof growth kinetics in cell culture for a large number of malignant celltypes originating from various source neoplasms. PV1(RIPO) unfoldedoncolytic activity in cell culture against HTB-14 and HTB-15 (FIG. 9),SF-767, SF-763, SF-295, and SF-188 glioblastoma cell lines (FIG. 10),HTB-185 medulloblastoma cell line (FIG. 11), CRL-7721 mammary carcinomacell line (FIG. 12), CRL-1435 and HTB-81 prostate carcinoma cell lines(FIG. 13), CCL-230 colon carcinoma cell line (FIG. 14), Hep-G2 and HuH7hepatocellular carcinoma cell lines (FIG. 15), CRL-2195 bronchialcarcinoma cell line (FIG. 16), and HEp-2 and HTB-32 epidermoid carcinomacell lines (FIG. 17). The growth characteristics of PV1(RIPO) in thispanel of malignant cell lines is representative for all PV/HRV2 IRESchimeras described in this application (data not shown).

EXAMPLE 5 Oncolysis of Astrocytomas with Chimeric Polioviruses

[0213] Using the astrocytoma cell lines HTB-14 and HTB-15 (obtained fromATCC), malignant gliomas were established through subcutaneous (HTB-15)and intracerebral (HTB-14) implantation of cells into nude mice. Thirtydays after inoculation of 5×10⁶ tumor cells into both subaxillary foldsof Cr:(NCr)-nufBR homozygotic nude mice, 70% of treated mice developedbilateral tumor growths that exceeded 6 mm in diameter. A group of 4mice with bilateral subcutaneous growths of at least 6 mm diameter wastreated by monolateral intraneoplastic inoculation of 10⁸ plaque formingunits (pfu) of PV1(RIPO). Tumor progression in the virus-treated groupor untreated controls, was followed clinically as well aspathologically. Whereas, tumor growth in untreated mice proceeded at asteady rate, yielding tumors above 9 mm diameter at 60 days postimplantation, malignant growths in the treated group recededdramatically. FIGS. 18A, 18C, and 18E show tumor tissue from anuntreated animal; FIGS. 18B, 18D and 18F show a receding neoplasm from avirus-injected mouse. Drastic shrinkage of tumor mass occurred uponvirus treatment (FIG. 18B) resulting in the formation of a wall ofnecrotic debris surrounding remaining tumor (FIG. 18D) that is beinginfiltrated by invading macrophages (FIG. 18F)]. After 14 days, tumorsin virus-treated mice could no longer be recognized by macroscopicobservation. Tumor recession was not limited to the neoplasm treatedwith intraneoplastic virus inoculation but also led to disappearance ofthe contralateral growth as judged by pathological analysis.Observations indicate that hematogenous spread of virus occurs afterintraneoplastic inoculation and releases amounts of virus sufficient toinfect and destroy tumors at distant sites.

[0214] The observation that i.v. administration of virus was sufficientfor maximal oncolysis was confirmed as follows: mice harboring growingtumors (>8 mm diameter) were infected iv with 5×10⁷ pfu PV1(RIPO). Tumorregress was assessed by weighing tumors in individual mice, sacrificedeach consecutive day following virus inoculation. See FIG. 19, wherein,grey bars indicate tumor weight, and intraneoplastic and extratumoralvirus replication is indicated by superimposed graphs. Tumors werehomogenized and the viral load was determined in a plaque assay. Drasticreduction in tumor size was accompanied by high levels of virusreplication within the receding neoplasm. Treatment of intracerebralgliomas with PV1(RIPO) led to tumor regress and remission. Mice receivedstereotactic intracerebral implants of 5×10⁴ HTB-14 cells.

[0215] Four groups, each comprising six mice harboring intracerebralgliomas, were formed. Group 1 was left untreated, group 2 received asingle intramuscular (i.m.) inoculation of 5×10⁷ pfu PV1(RIPO), group 3was administered a single intravenous (i.v.) inoculation of 5×10⁷ pfuPV1(RIPO), and group 4 received 5×10⁷ pfu PV1(RIPO) intracerebrally(i.c.). As can be seen in FIG. 20A, untreated mice succumbed toneurological complications stemming from the expanding intrahemisphericneoplasm 21-29 days following tumor implantation (average survival was26 days following tumor implantation). Mice treated with an i.m.inoculation of PV1(RIPO) had a slightly elevated life expectancy(average 40 days). In contrast, mice that had received i.v. inoculationof PV1(RIPO) had a significantly improved outcome of neoplastic disease(only 2 out of 6 mice died in consequence to tumor implantation; FIG.20C). Mice treated with a single i.c. inoculation of PV1(RIPO) werecompletely protected against malignant glioma (none of the treated micesuccumbed to their malignancy).

[0216] Occasionally, the athymic mice treated with an i.c. inoculationof PV1(RIPO) 12 days after tumor implantation experienced the emergenceof neurological symptoms in a period of 15-21 days post tumorimplantation (3-9 days following virus administration). However, evensevere symptoms of necrological dysfunction in these i.c. treated miceimproved within 1 week of virus administration. Most astonishingly, alltreated mice experienced complete recovery from their symptoms.Pathological analysis revealed that gliomas in untreated mice had grownto sizeable proportions accounting for the fatal outcome. See FIGS.21A-21E. FIGS. 21C and 21D show rapidly expanding tumor massesdistributed within the lateral ventricles; FIGS. 21A and 21B showcontrol sections from healthy mice. In contrast, FIG. 21E shows thatgliomas in treated mice underwent drastic shrinkage and eventualremission. In FIG. 21E a brain section, obtained from an animal treatedwith PV1(RIPO) 14 days after tumor implantation, shows the remnant of animplanted glioma leaving a tissue defect within brain parenchymabordering the left lateral ventricle.

[0217]FIGS. 22A to 22C show details from brain sections in FIG. 21. Acontrol section (FIG. 22A) shows the normal lateral ventricle with itsintact ependymal lining. FIG. 22B clearly shows a section through thebrain of an untreated mouse with tumor implant with a circular tumormass infiltrating the adjacent parenchyma. FIG. 22C shows a section ofthe brain of a virus-treated mouse with tumor implant. Macrophagicinfiltrates indicate removal of remaining debris stemming from anintraventricular neoplasm destroyed by PV1(RIPO).

EXAMPLE 6 Construction of PV1(prr)

[0218] An example for the construction of similar intradomain IRESchimeras is given for the generation of PV1(prr). PV1(prr) was producedby ligating a PCR product corresponding to PV1(M) IRES domains II-V(ascending loop) with the upper loop region of domain V (nt #492-508) ofHRV2 using primers (SEQ ID NO:24) and 5′-GGTTACGTGCTCTAGCTCCGAGGTTGGG-3′(SEQ ID NO:27) to a PCR fragment encompassing PV1(M) domain V(descending loop) using primers5′-AGAGCACGTAACCCAATGTGTATCTAGTCGTAACGCGCAACTCC-3′ (SEQ ID NO:28) and(SEQ ID NO:20) and a PCR fragment corresponding to PV1(M) domain VI withthe upper loop region (nt #582-609) of HRV2 using primers (SEQ ID NO:21)and (SEQ ID NO:2).

[0219] Recombinant IRES elements of various composition can be clonedinto the PV1(RIPO) cloning cassette and used to produce chimeric virusesby the methods described above.

[0220] PV1(prr) may be genotypically represented as: 5′cloverleaf(PV1)-IRES nt #106-484 (PV1)-IRES nt #484-508 (HRV2)-IRES nt #508-593(PV1)-IRES nt #594-612 (HRV2)-P1 [optionally derived from PV1/PV2/PV3 orPV1(S)/PV2(S)/PV3(S)]-P2 (PV1)-P3 excl. 3D^(pol) (nt #5111-5986; PV1)3D^(pol) [nt #5987-7369; PV1(S)]-3′NTR [optionally derived fromPV1/PV2/PV3 or PV1(S)/PV2(S)/PV3(S)]-poly(A) (PV1).

[0221] The nonpathogenic phenotype for PV1(prr) has been confirmed inone-step growth curves in SK-N-MC neuroblastoma cells (FIG. 8) and inCD155 tg mice (FIG. 8). Furthermore, PV1(prr) shares the oncolyticpotential of PV1(RIPO) in the panel of malignant cell lines described onpg. 48-49. Thus, the phenotypical hallmarks of PV1(RIPO) and PV1(RIPOS),lack of neuropathogenicity in cell culture and experimental animals withoncolytic potential against cultured malignant cell types, are alsoassociated with PV1(prr).

[0222] ccgaattcag aagcatgact ccaacgg 27

[0223]2) INFORMATION FOR SEQ. ID NO:8:

[0224] (i) SEQUENCE CHARACTERISTICS:

[0225] (A) LENGTH: 32

[0226] (B) TYPE: nucleotides

[0227] (C) STRANDEDNESS: single

[0228] (D) TOPOLOGY: linear

[0229] (ii) MOLECULE TYPE: cDNA

[0230] (iii) HYPOTHETICAL: No

[0231] (vi) ORIGINAL SOURCE:

[0232] (A) ORGANISM: Human Echovirus

[0233] (B) STRAIN: serotype 9

[0234] (viii) POSITION IN GENOME:

[0235] (A) CHROMOSOME/SEGMENT: IRES

[0236] (xi) SEQUENCE DESCRIPTION: SEQ. ID NO:8: gggagctccc attttgatgtattgagtgtt aa 32

[0237] (2) INFORMATION FOR SEQ. ID NO:9:

[0238] (i) SEQUENCE CHARACTERISTICS:

[0239] (A) LENGTH: 22

[0240] (B) TYPE: nucleotides

[0241] (C) STRANDEDNESS: single

[0242] (D) TOPOLOGY: linear

[0243] (ii) MOLECULE TYPE: cDNA

[0244] (iii) HYPOTHETICAL: No

[0245] (vi) ORIGINAL SOURCE:

[0246] (A) ORGANISM: Poliovirus

[0247] (B) STRAIN: Type 1 (Mahoney) (viii) POSITION IN GENOME:

[0248] (A) CHROMOSOME/SEGMENT: P1

[0249] (xi) SEQUENCE DESCRIPTION: SEQ. ID NO:9: ccgagctcag gtttcatcac ag22

[0250]2) INFORMATION FOR SEQ. ID NO:10:

[0251] (i) SEQUENCE CHARACTERISTICS:

1 28 1 30 DNA Poliovirus Type 1 Mahoney 1 ccgaattcaa cttagaagtttttcacaaag 30 2 30 DNA Human rhinovirus 14 2 cctagactcc catggtgccaatatatatat 30 3 27 DNA Human rhinovirus 14 3 ccggaattcc cacccatgaaacgttag 27 4 26 DNA Coxsackievirus 4 cctgagctcc atgatcacag tatatg 26 536 DNA Coxsackievirus 5 cttagaattc aaagaaacaa tggtcaatta ctgacg 36 6 20DNA Human echovirus 9 6 cctgagctcc cattttatcg 20 7 27 DNA Humanechovirus 9 7 ccgaattcag aagcatgact ccaacgg 27 8 30 DNA Poliovirus Type1 Mahoney 8 gggagctccc attttgatgt attgagtgtt 30 9 22 DNA Poliovirus Type1 Mahoney 9 ccgagctcag gtttcatcac ag 22 10 22 DNA Poliovirus Type 1Mahoney 10 ccgagctcag gtttcatcac ag 22 11 29 DNA Poliovirus Type 1Mahoney 11 ggagatcttg gatgccaaag cgctcgaag 29 12 25 DNA Poliovirus Type1 Mahoney 12 ggctcgagct tggttttgga cgggg 25 13 39 DNA Poliovirus Type 1Mahoney 13 ggctcgagcc cagtgctttc cactatgtgt ttgaagggg 39 14 28 DNAPoliovirus Type 1 Mahoney 14 tccggagcaa taaagctctt ccaattgg 28 15 53 DNAPoliovirus Type 1 Mahoney 15 gtccggagta ctcaacattg taccgccgtt ggcttgactcattttagtaa ccc 53 16 26 DNA Human rhinovirus 2 16 ggtgcgaacg ttgttgccattgctgc 26 17 26 DNA Poliovirus Type 1 Mahoney 17 ccggatccaa agcgagcacacggggc 26 18 25 DNA Poliovirus Type 1 Mahoney 18 ccggatcctc cggcccctgaatgcg 25 19 30 DNA Human rhinovirus 2 19 cctgagctcc cattatgatacaattgtctg 30 20 33 DNA Human rhinovirus 2 20 ggtaccaata aaataaaaggaaacacggac acc 33 21 29 DNA Human rhinovirus 2 21 gcggtaccgc ttatggtgacaatatatac 29 22 27 DNA Human echovirus 9 22 ccggtaccta aaggaaaaagtgaaaca 27 23 28 DNA Human echovirus 9 23 ccggtaccgc ttatggtgac aatcacag28 24 25 DNA Human rhinovirus 2 24 gggaattcag acgcacaaaa ccaag 25 25 25DNA Human rhinovirus 2 25 ccggatcctt atgtagctca atagg 25 26 25 DNA Humanrhinovirus 2 26 ccggatcctt atgtagctca atagg 25 27 28 DNA Humanrhinovirus 2 27 ggttacgtgc tctagctccg aggttggg 28 28 44 DNA Humanrhinovirus 2 28 agagcacgta acccaatgtg tatctagtcg taacgcgcaa ctcc 44

What we claim are:
 1. A recombinant poliovirus constructed from apoliovirus having a 5′NTR region containing an internal ribosomal entrysite (IRES), and the coding sequences for structural proteins (P1), andfor the non-structural proteins (P2 and P3) and a 3′NTR selected fromthe group consisting of wild type serotype 1, serotype 2, and serotype3, wherein a. i. a part of the IRES of the poliovirus is substitutedwith a part of the IRES of Human Rhinovirus serotype 2 also having a5′NTR region containing an internal ribosomal entry site (IRES), thecoding sequences of structural proteins (P1), and for the non-structuralproteins (P2 and P3) and a 3′NTR, or ii. at least a part of the IRES ofthe poliovirus is substituted with at least a part of the IRES of avirus selected from the group of picornaviruses comprising HumanRhinovirus serotype 1-100, coxsackievirus serotype B1-B6, humanechovirus serotype 1-7, 9, 11-27, and 29-33, also having a 5′NTR regioncontaining an internal ribosomal entry site (IRES), the coding sequencesof structural proteins (P1), and for the non-structural proteins (P2 andP3) and a 3′NTR, and wherein b. optionally, at least a part of the P1 ofthe poliovirus is substituted with at least a part of the P1 of aPoliovirus (Sabin), selected from the groups consisting of PV1(S),PV2(S) and PV3(S); c. optionally, at least a part of the P3 of thepoliovirus is substituted with at least a part of the P3 of a Poliovirus(Sabin), selected from the groups consisting of PV1(S), PV2(S) andPV3(S); and d. optionally, at least a part of the 3′NTR of thepoliovirus is substituted with at least a part of the 3′NTR of aPoliovirus (Sabin), selected from the group consisting of PV1(S),PV2(S), and PV3(S).
 2. A recombinant poliovirus according to claim 1wherein a part of the IRES of the poliovirus is substituted with a partof the IRES of the Human Rhinovirus serotype
 2. 3. A recombinantpoliovirus according to claim 1 wherein at least a part of the IRES ofthe poliovirus is substituted with at least a part of the IRES of theHuman Rhinovirus serotype 1, 3-100.
 4. A recombinant poliovirusaccording to claim 2 wherein at least a part of the IRES of thepoliovirus is substituted with at least a part of the IRES of the HumanRhinovirus serotype
 14. 5. A recombinant poliovirus according to claim 1wherein at least a part of the IRES of the poliovirus is substitutedwith at least a part of the IRES of the coxsackievirus serotype B1 toB6.
 6. A recombinant poliovirus according to claim 5 wherein at least apart of the IRES of the poliovirus is substituted with at least a partof the IRES of the coxsackievirus serotype B4.
 7. A recombinantpoliovirus according to claim 1 wherein at least a part of the IRES ofthe poliovirus is substituted with at least a part of the IRES of thehuman echovirus serotype 1 to 7, 9, 11 to 27, and 29 to
 33. 8. Arecombinant poliovirus according to claim 7 wherein at least a part ofthe IRES of the poliovirus is substituted with at least a part of theIRES of the human echovirus serotype
 9. 9. A recombinant poliovirusPV1(R2-4,6) according to claim 3 wherein the poliovirus is PV1(M) andthe IRES domains II, III, IV and VI are substituted with the IRESdomains II, III, IV and VI of the Human Rhinovirus serotype
 2. 10. Arecombinant poliovirus PV1(R5) according to claim 3 wherein thepoliovirus is PV1(M) and the IRES domain V is substituted with the IRESdomain V of the Human Rhinovirus serotype
 2. 11. A recombinantpoliovirus PV1(R2-5) according to claim 3 wherein the poliovirus isPV1(M) and the IRES domains II, III, IV and V is substituted with theIRES domains II, III, IV and V of the Human Rhinovirus serotype
 2. 12. Arecombinant poliovirus PV1(R5-6) according to claim 3 wherein thepoliovirus is PV1(M) and the IRES domains V and VI is substituted withthe IRES domains V and VI of the Human Rhinovirus serotype
 2. 13. Arecombinant poliovirus PV1(R6) according to claim 3 wherein thepoliovirus is PV1(M) and the IRES domain VI is substituted with the IRESdomain VI of the Human Rhinovirus serotype
 2. 14. A recombinantpoliovirus PV1(prr) according to claim 3 wherein the poliovirus isPV1(M) and nt#484-nt#508 of the IRES domain V and nt#594-nt#612 of theIRES domain VI is substituted with nt#484-nt#508 of the IRES domain Vand nt#594-nt#612 the IRES domain VI of the Human Rhinovirus serotype 2.15. A composition comprising a recombinant poliovirus according to claim1 and a pharmaceutically acceptable carrier.
 16. A compositioncomprising a recombinant poliovirus according to claim 2 and apharmaceutically acceptable carrier.
 17. A composition comprising arecombinant poliovirus according to claim 3 and a pharmaceuticallyacceptable carrier.
 18. A composition comprising a recombinantpoliovirus according to claim 4 and a pharmaceutically acceptablecarrier.
 19. A composition comprising a recombinant poliovirus accordingto claim 5 and a pharmaceutically acceptable carrier.
 20. A compositioncomprising a recombinant poliovirus according to claim 6 and apharmaceutically acceptable carrier.
 21. A composition comprising arecombinant poliovirus according to claim 7 and a pharmaceuticallyacceptable carrier.
 22. A composition comprising a recombinantpoliovirus according to claim 8 and a pharmaceutically acceptablecarrier.
 23. A composition comprising a recombinant poliovirus accordingto claim 9 and a pharmaceutically acceptable carrier.
 24. A compositioncomprising a recombinant poliovirus according to claim 10 and apharmaceutically acceptable carrier.
 25. A composition comprising arecombinant poliovirus according to claim 11 and a pharmaceuticallyacceptable carrier.
 26. A composition comprising a recombinantpoliovirus according to claim 12 and a pharmaceutically acceptablecarrier.
 27. A composition comprising a recombinant poliovirus accordingto claim 13 and a pharmaceutically acceptable carrier.
 28. A compositioncomprising a recombinant poliovirus according to claim 14 and apharmaceutically acceptable carrier.
 29. A composition according to anyone of claims 15-28 wherein the composition is infusible.
 30. Acomposition according to any one of claims 15-28 wherein the compositionis indictable.
 31. A composition according to any one of claims 15-28wherein the pharmaceutically acceptable carrier is a physiological saltsolution.
 32. A composition according to any one of claims 15-28 whereinthe physiological salt solution is HANKS balanced salt solution.
 33. Atherapeutic method of treating malignant tumors comprising: A. Preparinga recombinant poliovirus from a poliovirus having a 5′ NTR regioncontaining an internal ribosomal entry site (IRES), and the codingsequences for structural proteins (P1), and for the non-structuralproteins (P2 and P3) and a 3′NTR selected from the group consisting ofwild type serotype 1, serotype 2, and serotype 3, by a. substituting atleast a part of the IRES of the poliovirus with at least ap art of theIRES of a virus selected from the group of picornaviruses comprisingHuman Rhinovirus serotype 1, 3-100, coxsackievirus serotype B1-B6, humanechovirus serotype 1-7, 9, 11-27, 29-33, also having a 5′NTR regioncontaining an internal ribosomal entry site (IRES), the coding sequencesof structural proteins (P1), and for the non-structural proteins (P2 andP3) and a 3′NTR, and wherein b. optionally, substituting at least a partof the P1 of the poliovirus with at least a part of the P1 of aPoliovirus (Sabin), selected from the groups consisting of PV1(S),PV2(S) and PV3(S); c. optionally, substituting at least a part of the P3of the poliovirus with at least a part of the P3 of a Poliovirus(Sabin), selected from the groups consisting of PV1(S), PV2(S) andPV3(S); d. optionally, substituting at least a part of the 3′NTR of thepoliovirus with at least a part of the 3′NTR of a Poliovirus (Sabin),selected from the group consisting of PV1(S), PV2(S), and PV3(S); and B.Administering directly to the tumor site, intrathecally or intravenouslya composition comprising the recombinant poliovirus.
 34. A therapeuticmethod of treating the malignant tumors according to claim 33 whereinthe recombinant virus is prepared by substituting at least a part of theIRES of the poliovirus with at least a part of the IRES of the HumanRhinovirus serotype 1 to
 100. 35. A therapeutic method of treatingmalignant tumors according to claim 33 wherein the recombinant virus isprepared by substituting at least a part of the IRES of the polioviruswith at least a part of the IRES of the Human Rhinovirus serotype 14.36. A therapeutic method of treating malignant tumors according to claim33 wherein the recombinant virus is prepared by substituting at least apart of the IRES of the poliovirus with at least a part of the IRES ofthe coxsackievirus serotype B1 to B6.
 37. A therapeutic method oftreating malignant tumors according to claim 33 wherein the recombinantvirus is prepared by substituting at least a part of the IRES of thepoliovirus with at least a part of the IRES of the coxsackievirusserotype B4.
 38. A therapeutic method of treating malignant tumorsaccording to claim 33 wherein the recombinant virus is prepared bysubstituting at least a part of the IRES of the poliovirus with at leasta part of the IRES of the human echovirus serotype 1 to 7, 9, 11 to 27,29 to
 33. 39. A therapeutic method of treating malignant tumorsaccording to claim 33 wherein the recombinant virus is prepared bysubstituting at least a part of the IRES of the poliovirus with at leasta part of the IRES of the human echovirus serotype
 9. 40. A therapeuticmethod of treating malignant tumors according to claim 33 wherein therecombinant virus is PV1(R2-4,6) prepared from poliovirus PV1(M) and theIRES domains II, III, IV and VI thereof are substituted with the IRESdomains II, III, IV and VI of the Human Rhinovirus serotype
 2. 41. Atherapeutic method of treating malignant tumors according to claim 33wherein the recombinant virus is PV1(R5) prepared from poliovirus PV1(M)and the IRES domain V thereof is substituted with the IRES domain V ofthe Human Rhinovirus serotype
 2. 42. A therapeutic method of treatingmalignant tumors according to claim 33 wherein the recombinant virus isPV1(R2-5) prepared from poliovirus PV1(M) and the IRES domains II, III,IV and V thereof is substituted with the IRES domains II, III, IV and Vof the Human Rhinovirus serotype
 2. 43. A therapeutic method of treatingmalignant tumors according to claim 33 wherein the recombinant virus isPV1(R5-6) prepared from poliovirus PV1(M) and the IRES domains V and VIthereof is substituted with the IRES domains V and VI of the HumanRhinovirus serotype
 2. 44. A therapeutic method of treating malignanttumors according to claim 33 wherein the recombinant virus is PV1(R6)prepared from poliovirus PV1(M) and the IRES domain VI thereof issubstituted with the IRES domain VI of the Human Rhinovirus serotype 2.45. A therapeutic method of treating malignant tumors according to claim33 wherein the recombinant virus is PV1(prr) prepared from PV1(M) andnt#484-nt#508 of the IRES domain V and nt#594-nt#612 of the IRES domainVI is substituted with nt#484-nt#508 of the IRES domain V andnt#594-nt#612 the IRES domain VI of the Human Rhinovirus serotype
 2. 46.A therapeutic method of treating malignant tumors according to any oneof claims 33-45 wherein the malignant tumor is selected from a groupconsisting of glioblastoma multiforme, medulloblastoma, mammarycarcinoma, prostate carcinoma, colorectal carcinoma, hepatocellularcarcinoma, bronchial carcinoma, and epidermoid carcinoma.
 47. Atherapeutic method of treating malignant tumors according to claim 46wherein the malignant tumor is glioblastoma multiforme.
 48. Atherapeutic method of treating malignant tumors according to claim 46wherein the malignant tumor is medulloblastoma.
 49. A therapeutic methodof treating malignant tumors according to claim 46 wherein the malignanttumor is mammary carcinoma.
 50. A therapeutic method of treatingmalignant tumors according to claim 46 wherein the malignant tumor isprostate carcinoma.
 51. A therapeutic method of treating malignanttumors according to claim 46wherein the malignant tumor is colorectalcarcinoma.
 52. A therapeutic method of treating malignant tumorsaccording to claim 46 wherein the malignant tumor is hepatocellularcarcinoma.
 53. A therapeutic method of treating malignant tumorsaccording to claim 46 wherein the malignant tumor is bronchialcarcinoma.
 54. A therapeutic method of treating malignant tumorsaccording to claim 46 wherein the malignant tumor is epidermoidcarcinoma.
 55. A therapeutic method of treating malignant tumorsaccording to any one of claims 33-45 wherein the route of administrationis intravenous.
 56. A therapeutic method of treating malignant tumorsaccording to any one of claims 33-45 wherein the route of administrationis intrathecal.
 57. A therapeutic method of treating malignant tumorsaccording to any one of claims 33-45wherein the route of administrationis directly to the tumor site.